Methods for treating cancer

ABSTRACT

The present invention is directed towards the diagnosis of malignant cancer by detection of the mts-1 MRNA or the  mts-1  protein, encoded by the  mts-1  gene. The present invention contemplates the use of recombinant mts-1 DNA and antibodies directed against the mts-1 protein to diagnose the metastatic potential of several types of tumor cells, including, for example, thyroid, epithelial, lung, liver and kidney tumor cells. The present invention is also directed to mammalian cell lines and tumors with high and low metastatic potential which have been developed to serve as tseful model systems for in vitro and in vivo anti-metastasis drug screening.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation-In-Part of U.S. Ser. No. 08/468,942 filed Jun. 6,1995 now U.S. Pat. No. 5,965,360, which is a Divisional of U.S. Ser. No.08/190,560 filed Jan. 31, 1994 now U.S. Pat. No. 5,798,257, which is aContinuation-In-Part of U.S. Ser. No. 07/981,455 filed Nov. 25, 1992 andnow abandoned, which is a Rule 60 Continuation of U.S. Ser. No.07/550,600 filed Jul. 9, 1990 now abandoned.

FIELD OF THE INVENTION

The present invention is directed towards the diagnosis of malignantcancer by detection of the mts-1 mRNA or the mts-1 protein encoded bythe mts-1 gene. This invention further relates to cancer therapeutics.More particularly, this invention relates to compositions and methodsfor treating tumors, for example, by intercepting the interactionbetween Mts-1 and p53. The present invention is also directed tomammalian cell lines and tumors with high and low metastatic potentialwhich have been developed to serve as useful model systems for in vitroand in vivo anti-metastasis drug screening.

BACKGROUND OF THE INVENTION

Malignant cancer tumors shed cells which migrate to new tissues andcreate secondary tumors; a benign tumor does not generate secondarytumors. The process of generating secondary tumors is called metastasisand is a complex process in which tumor cells colonize sites distantfrom the primary tumor. Tumor metastasis remains the major cause ofmorbidity and death for patients with cancer. One of the greatestchallenges in cancer research is to understand the basis of metastasis,i.e., what controls the spread of tumor cells through the blood andlymphatic systems and what allows tumor cells to populate and flourishin new locations.

The metastatic process appears to be sequential and selective, and iscontrolled by a series of steps since metastatic tumor cells: (a) aremobile and can disseminate from the original tumor; (b) are capable ofinvading the cellular matrix and penetrating through blood vessels; (c)possess ummunological markers, which allow them to survive passagethrough the blood stream, where they must avoid the immunologicallyactive cytotoxic “T” lymphocytes; and (d) have the ability to find afavorable location to transplant themselves and successfully survive andgrow.

Understanding the underlying molecular mechanisms in metastasis is thekey to understanding cancer biology and its therapy. In clinicallesions, malignant tumors contain a heterogeneous population of cells,exhibiting a variety of biological characteristics, e.g., differentialgrowth rates, cell surface structures, invasive capacities andsensitivity to various cytotoxic drugs. Researchers can take advantageof tumor heterogeneity factors, by identifying specific cell producedmarkers, which are unique for metastasis, to develop therapeuticregiments which do not rely only on surgical resection.

At this time it is not known whether the metastatic phenotype is underthe regulation of a single or multiple gene(s), and whether these genesare independent or interrelated. However, a number of genes have becomecorrelated with the formation and metastasis of tumors. For example,several normal cellular genes become oncogenes by incorporation into aretroviral genome. Due to the juxtaposition of new promoter elements,such incorporation frequently allows a potential oncogene to beexpressed in inappropriate tissues or at higher levels than it normallywould be expressed. It appears from work with tumorigenic retrovirusesas well as other systems that misexpression of many cellular proteins,particularly those involved in the regulation of the cell cycle, cellmobility, or cell-cell interaction may lead to a cancerous phenotype.

The present invention discloses the human mts-1 gene and diagnosis ofmetastatic cancer by use of either antibodies directed against the mts-1protein or mts-1 nucleic acid probes directed against mts-1 mRNA.

The mouse and rat mts-1 genes have been previously isolated underdifferent names (i.e., 18A2, Linzer, et al., Proc. Natl. Acad. Sci. USA.80:4271-4275, 1983; and p9Ka, Barraclough et al., J. Mol. Biol.198:13-20, 1987) but no function or correlation of the mts-1 gene inmetastatic cancer has been established prior to the present invention.Previous work has indicated that the protein now identified as the mts-1protein is a calcium binding protein with homology to other calciumbinding proteins such as, for example, the S-100 calcium protein, whichare thought to have a role in cell growth (Linzer et al. supra;Jackson-Grusby et al., Nuc. Acids Res. 15:6677-6690, 1987; Goto et al.,J. Biochem. 103:48-53, 1988). Other researchers suggest a role for p9Ka,later found to be identical to mts-1, in myoepithelial celldifferentiation (Barraclough, et al., supra).

As determined uniquely by the present invention, the mammalian mts-1gene is expressed at 10-100 fold higher levels in metastatic cellscompared to non-metastatic cells and normal cells. Only a few types ofnormal cells, including lymphocytes and trophoblasts, express mts-1.Hence, the present invention demonstrates a surprising new property ofmts-1: the misexpression of mts-1 within a cell or tissue is diagnosticof malignant cancer.

p53 is a tumor suppressor protein found in humans and other mammals(See, e.g., Harris, Science 262: 1980-1981, 1993). The wild-type p53protein functions to regulate cell proliferation and cell death (alsoknown as apoptosis). While the mechanism through which the wild-type p53protein suppresses tumor cell growth is not completely defined, it isknown that one key feature of the growth suppression is the capacity ofp53 to act as a transcription factor (Farmer et al., Nature 358, 83-86,1992; and Kern et al., Science 256, 827-830, 1992).

The nucleotide and amino acid sequences of human p53 have been reportedby Zakut-Houri et al, EMBO J. 4: 1251-1255, 1985). The ability of p53 tobind DNA in a sequence-specific manner maps to amino acid residues90-290 of human p53 (Pavletich et al, Genes Dev. 7: 2556-2564, 1993; andWang et al, Genes Dev. 7: 2575-2586 1993); the tetramerization domainmaps to amino acid residues 322-355 of human p53. The DNAbinding-regulation domain maps to amino acid residues 364-393 of humanp53 or to the corresponding region encompassing residues 361-390 ofmouse p53 (Hupp et al., Cell 71: 875-886, 1992; and Halazonetis et al.,EMBO J. 12: 1021-1028, 1993).

Inactivation of p53 is associated with more than half of all humantumors. The inactivation can occur by mutation of the p53 gene orthrough binding of p53 to viral or cellular oncogene proteins, such asthe SV40 large T antigen and MDM2. Mutations of the p53 protein in mosthuman tumors involve the sequence-specific DNA binding domain(Bargonetti et al., Genes Dev. 6: 1886-1898, 1992).

The present invention has further identified the tumor suppressorprotein p53 as a target for the metastasis associated Mts1 protein.

SUMMARY OF THE INVENTION

The present invention is directed towards the diagnosis of metastaticcancer using an mts-1 nucleic acid or antibodies directed against themts-1 protein. The present invention is also directed to isolated andpurified mts-1 nucleic acids available for diagnostic tests andantibodies directed against the mammalian mts-1 proteins.

One aspect of the present invention is directed to a method fordiagnosing metastatic cancer by contacting serum from an individual tobe tested for such cancer with an antibody reactive with a mammalianmts-1 protein or an antigenic fragment thereof, for a time and underconditions sufficient to form an antigen-antibody complex, and detectingthe antigen-antibody complex.

Another aspect of the present invention provides an isolated,recombinant nucleic acid encoding a human mts-1 gene or a fragmentthereof, and replicable DNA sequences encoding an mts-1 polypeptidewhich express high levels of the mts-1 polypeptide. Isolated antisensemts-1 nucleic acids and expression vectors therefor are alsocontemplated by the present invention. Human mts-1 nucleic acids arepreferred.

A further aspect of this invention is directed to isolated transformedhost cells, such as prokaryotic microorganisms, yeast, insect cells andeukaryotic cells, containing mts-1 nucleic acids and replicable vectorscontaining DNA sequences encoding the mts-1 polypeptide.

A still further aspect of this invention provides isolated homogeneousmammalian mts-1 polypeptides and pharmaceutical compositions includingsuch a mts-1 polypeptide or protein. Human mts-1 polypeptides arepreferred.

Another aspect of this invention provides antibodies directed against anmts-1 polypeptide or any peptide, fragment or derivative of the mts-1protein.

A further aspect of this invention is directed towards treatment ofcancer by administering reagents, such as for example, anti-mts-1antibodies capable of binding the mts-1 protein and antisense mts-1nucleic acids capable of binding mts-1 sense mRNA.

One aspect of the invention provides compounds which interfere with theinteraction between Mts-1 and p53 by binding to Mts-1 (i.e.,binding-intercepting compounds).

Another embodiment of the present invention provides a method forintercepting the binding between p53 and Mts-1 in a subject byadministering to the subject, an effective amount of a peptide whichprevents the interaction between p53 and Mts-1 by binding to Mts-1. Forexample, one such peptide comprises the C-terminal region of p53 (aminoacid 289-393 of human p53 or amino acid 289-390 of murine p53), inparticular, amino acid 360-393 of human p53 or amino acid 360-390 ofmurine p53. Functional fragments or analogs of such peptides are alsowithin the scope of the present invention. Another example of abinding-intercepting peptide comprises amino acid 1909-1937 ofnon-muscle myosin heavy chain or functional fragments of analogsthereof.

In one embodiment, the present invention provides methods of treating atumor in a subject by administering to the subject, a therapeuticallyeffective amount of a nucleic acid molecule coding for a peptide whichprevents the binding of Mts-1 to p53.

In one embodiment, the present invention provides methods of treating atumor in a subject by administering to the subject, a therapeuticallyeffective amount of a peptide which prevents the binding of Mts-1 top53.

In another embodiment, the present invention provides a method oftreating a tumor in a subject by administering to the subject, atherapeutically effective amount of an antibody directed against Mts-1.

In still another embodiment, the present invention provides methods oftreating a tumor in a subject by administering a therapeuticallyeffective amount of an antisense DNA of an Mts-1 gene.

Yet another aspect of the present invention provides an animal modelsystem of the metastatic process, including several eukaryotic celllines and tumors expressing different levels of mts-1, which are derivedfrom mouse and rat carcinomas. These cell lines and tumors may bere-introduced into mice or rats to produce primary tumors whichmetastasize to the lung, liver and kidneys with a characteristicfrequency. Therefore, the present invention also provides a wellcontrolled animal model system for testing pharmaceutical compositionssuspected to have therapeutic utility for the treatment of metastaticcancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 depicts the nucleotide sequence of the coding region of the humanmts-1 gene.

FIG. 2 depicts the amino acid sequence of the human mts-1 protein.

FIG. 3 depicts the circular, expression plasmid pEMSVscribe2 containingthe complete coding region of mts-1 under the control of the murinesarcoma virus promoter (MSV LTR).

FIG. 4 illustrates an autoradiograph showing detection of the mts-1transcript by a mts-1 nucleic acid probe in a Northern blot of mRNA froma cell line with low metastatic potential (CSML-0) and a cell line withvery high metastatic potential (CSML-100).

FIG. 5 illustrates an autoradiograph showing detection of the mts-1transcript by a mts-1 nucleic acid probe in a Northern blot of mRNA fromdifferent metastatic (depicted with an “M” above the lane) andnon-metastatic mouse tumors and cell lines. In the top autoradiograph;Lane 1-HMC-Lr; Lane 2-HMC-0; Lane 3-RL-67; Lane 4-B-16, Lane 5-LLC; Lane6-Acatol; Lane 7-C12; Lane 8-PCC4c-B; Lane 9-PCC4c-P, Lane 10-PCC4c-107;Lane 11-PCC4107; Lane 12-T9; Lane 13-LMEC; Lane 14-T36; Lane 15-T36cL.The bottom autoradiograph depicts the same Northern blot hybridized withan actin probe, providing a comparison of the amounts of mRNA in eachlane.

FIG. 6 illustrates an autoradiograph showing detection of the mts-1transcript by a mts-1 nucleic acid probe in a Northern blot of mRNA fromvarious tumors and tumor cell lines. Lanes 1 and 2-size markers; Lane3-mouse lung carcinoma Line 1 grown without DMSO; Lane 4-mouse lungcarcinoma Line 1 grown with 3% DMSO; Lane 5-IR6 tumor; Lane 6-TRCL₁ cellline; Lane 7-IR6 cell line (IR6CL₁); Lane 8-FRTL5 cell line.

FIG. 7 depicts a histopathological characterization of some of the rattumors of the present invention, demonstrating the morphological andhistological identity of these tumors with corresponding human tumors.

FIG. 8 illustrates an autoradiograph showing detection of the mts-1transcript by a mts-1 nucleic acid probe in a Northern blot of mRNA fromvarious Line 1 murine lung carcinoma cell lines containing a transfectedcopy of the rat mts-1 gene (N1-N10), or just an antibiotic resistancemarker (Neo 1-3), all grown in the present of 3% DMSO; compared to Line1 cells grown without DMSO (Line 1). DMSO inhibits the development ofthe metastatic phenotype as well as mts-1 expression in non-transfectedLine 1 cells, hence transfection of mts-1 can overcome this block.

FIG. 9a depicts the lungs from 3 mice injected subcutaneously with 1×10⁶CSML-0 cells. Lungs were removed 4-6 weeks after injection and theninjected with India ink. Dark areas indicate normal tissues; white areasare tumors.

FIG. 9b depicts the lungs from 3 mice injected intravenously with 1×10⁴CSML-0 cells. Lungs were removed 15 days after injection and theninjected with India ink. Dark areas indicate normal tissues; white areasare tumors.

FIG. 9c depicts the lungs from 3 mice injected intravenously with 1×10⁴CSML-100 cells. Lungs were removed 15 days after injection and theninjected with India ink. Dark areas indicate normal tissues; white areasare tumors.

FIG. 9d depicts the lungs from 3 mice injected subcutaneously with 1×10⁶CSML-100 cells. Lungs were removed 4-6 weeks after injection and theninjected with India ink. Dark areas indicate normal tissues; white areasare tumors.

FIG. 9e depicts the lungs from 3 mice injected with 0.1 ml serum-freemedia. Lungs were removed 6-8 weeks after injection and then injectedwith India ink. Dark areas indicate normal tissues; white areas aretumors.

FIG. 10a depicts a diagram of the more important regions of the pTrcHisB expression vector utilized to produce a histidine-mts-1 fusionprotein. The murine mts-1 cDNA was subcloned into pTrcHis B at theBamHI-KpnI site to generate pTBM1.

FIG. 10b depicts a Coomassie Brilliant Blue-stained gel illustrating theprofile of proteins eluted from a Ni⁺⁺-NTA column used to purify mts-1protein expressed by cells containing pTBM1. Elution was with a seriesof buffers having pH values varying from 5.9 to 4.5. A single majorprotein, the mts-1 protein, is eluted.

FIG. 11 depicts a growth curve of CSML-0 and CSML-100 cells over a fiveday period. Cell growth was measured daily by observing the number ofcells per dish (ordinate). As illustrated, CSML-100 cells, which expresshigh levels of mts-1, grow at a slower rate than CSML-0 cells whichexpress little mts-1.

FIG. 12a depicts a photomicrograph of a section from an 8 day mouseembryo hybridized with a ³H-labelled mts-1 antisense probe. Signal isdetected in the trophoblast cells.

FIG. 12b depicts a photomicrograph of a section from an 8 day mouseembryo hybridized with a ³H-labelled mts-1 sense probe. No signal isdetected.

FIG. 13 depicts a western blot of CSML-0 (Lane 1) and CSML-100 (Lanes 2and 3) cell lysates. Lanes 1 and 2 were probed with the chickenanti-mts-1 antibody (α-mts-1) using a secondary antibody (rabbitanti-chicken IgG-HRP) for detection. Lane 3 was similarly probed exceptthat free mts-1 protein was added during the incubation with the α-mts-1antibody. An approximate 10-12. kd mts-1 protein is detected only inCSML-100 cells and only when no free mts-1 protein is present to competefor binding to the α-mts-1 antibody. Therefore, the α-mts-1 antibody ishighly specific for mts-1 protein.

FIG. 14a depicts a frozen mouse spleen section probed with the α-mts-1antibody. Rabbit anti-chicken IgG-HRP was used for detection of themts-1 antigen-antigen complex (dark spots).

FIG. 14b depicts a frozen mouse spleen section probed with the α-mts-1antibody in the presence of free mts-1 protein. Rabbit anti-chickenIgG-HRP was used for detection of the mts-1 antigen-antigen complex(dark spots). As illustrated, little or no mts-1 protein is detectedwhen free mts-1 protein is present to compete for binding to α-mts-1(compare to FIG. 14a). Therefore, the α-mts-1 antibody is highlyspecific for mts-1 protein.

FIG. 15a illustrates that mts-1 protein can be detected only in serumfrom mice injected with CSML-100 cells. This figure depicts a westernblot of serum taken from non-injected mice (Lane 3), mice injected with1×10⁵ CSML-0 cells (Lane 1) and mice injected with 1×10⁵ CSML-100 cells(Lane 2). After reaction with the α-mts-1 antibody a 10-12 kd mts-1protein is detected only in the serum from mice injected with CSML-100cells. The higher molecular weight bands merely cross-react with theanti-mts-1 antibody used and were not mts-1 proteins.

FIG. 15b similarly illustrates that mts-1 protein can be detected onlyin serum from mice injected with CSML-100 cells. This figure depicts awestern blot of serum taken from non-injected mice (Lane 3), miceinjected with 1×10⁶ CSML-0 cells (Lane 1) and mice injected with 1×10⁶CSML-100 cells (Lane 2). After reaction with the α-mts-1 antibody a10-12 kd mts-1 protein is detected only in the serum from mice injectedwith CSML-100 cells. As described, the higher molecular weight bandsmerely cross-react with the anti-mts-1 antibody used and were not mts-1.

FIG. 15c depicts a western blot of lysed whole blood from mice probedwith the α-mts-1 antibody. Lanes 1-4 were loaded with 5, 10, 20 and 25μl lysed whole blood, respectively. Lane 5 was loaded with CSML-100 celllysate as a positive control. This blot illustrates that mts-1 proteinin serum is not simply due to lysis of lymphocyte or blood cells.

FIG. 15d depicts a western blot of increasing amounts of serum from miceinjected with salmonella lipopolysaccharide (LPS) to induce a chronicimmune response. The blot was probed with the α-mts-1 antibody to revealany detectable mts-1 protein. Lanes 1-3 were loaded with 75, 100 or 150μg serum, respectively. This blot illustrates that mts-1 protein inserum is not derived from activated macrophages generated by a chronicimmune response.

FIG. 16 depicts a western blot of sera from patients with non-metastaticand metastatic cancers probed with the α-mts-1 antibody to reveal anydetectable mts-1 protein. A 27 kd mts-1 protein is detected only inpatients known to have metastatic cancer. Sera were taken from patientswith non-metastatic breast cancer (Lane 1), with non-metastaticlymphomas (Lanes 2 and 4), with metastatic lymphomas (Lanes 5 and 7) andwith metastatic breast cancer (Lane 6). Lane 3 contains normal serum asa negative control. The higher molecular weight proteins merelycross-react with the α-mts-1 antibody and do not represent mts-1 proteinproducts.

FIG. 17A and FIG. 17B depict co-immunoprecipitation of Mts1 and p53.

Top panel—CSML-100 lysates immunoprecipitated by different batches ofanti-Mts1 antibody (1-4) and control antibody (C). Antibodies 3 and 4effectively pulled down Mts1-protein complexes with Myosin (200 kDa) andp53 (53 kDa). Antibodies 1 and 4 are less effective for complex IP.

Bottom panel—Cells were metabolically labelled with ³⁵S-Methionin andimmunoprecipitation was performed using: lysate from CSML-100cells+anti-Mts1 serum (lane 1), lysate from CSML-100 cells+controlantibody (lane 2), lysate from CSML-0 cells+pAb 421 (lane 3), lysatefrom CSML-100 cells+pAb 421 (lane 4).

FIG. 18 depicts immunoprecipitation of non-labeled Mts-1 from CSML-100cells using anti-p53 antibodies directed to various epitopes.

FIG. 19 depicts the interaction between Mts1 and the C-terminal domainof p53. Full size recombinant p53 and its domains were mixed withrecombinant Mts1 and pooled-down with anti-p53 antibodies. Western blotwas performed with immunoprecipitates followed by immunoprobing withanti-Mts1 antibody. Lanes 1,2-full size p53; 3,4-N-terminal domain;5,6-DNA-binding domain; 7,8-C-terminal domain; and in 1,3,5 and7-control antibody was used.

FIG. 20 depicts binding of the recombinant Mts1 to p53-GST fusionproteins fixed on Glutahione-sepharose beads. Mts1 associated with GST(negative control), GST- p53 (wild type p53) and GST-p53-Δ30 (mutatedp53 lacking amino acids 364-393) was analyzed by western blot withfollowing immunoprobing with anti-mts1 antibody.

FIG. 21 depicts Mts1 interaction with target proteins in a blot-overlayassay. Recombinant full size p53 (1), N-terminal domain (2), DNA-bindingdomain (3), C-terminal domain (4) and the fragment of the non-musclemyosin (5) after gel electrophoresis were transferred ontonitrocellulose membrane. Identical membranes were incubated withdifferent batches of the recombinant Mts1 protein (Mts1-a, Mts1-b,Mts1-c and Mts1-d). Mts1 bound to the fixed proteins was detected by theanti-Mts1 serum. The graph at the upper left depicts the schematiclocalization of the proteins on the membranes.

FIG. 22A and FIG. 22B depict the Mts-1 inhibition of p53 phosphorylationby PKC on the C-terminal domain in vitro.

Top—phosphorylation of full size recombinant p53 in the presence andabsence of recombinant Mts1.

Bottom—phosphorylation of the N-terminal domain (left), the DNA-bindingdomain (middle) and the C-terminal domain of P53 in presence of Mts1(with increasing Mts concentrations from left lanes to right lanes ineach radiography).

FIG. 23 depicts phosphorylation by CKII full size p53 (lanes 1,2) andC-terminal domain of p53 (lanes 3,4) in the absence (lanes 1,3) andpresence (lanes 2,4) of the recombinant Mts1. Left panel:autoradiography of the dried gel; right panel—Coomassie staining of thesame gel.

FIG. 24 depicts EMSA using CSML-0 nuclear extracts with p53 binding sitefrom p21/WAF1 promoter.

FIG. 25A depicts the inhibition by Mts1 of the p53 transactivation ofthe p21/WAF2-luciferase reporter gene in CSML-0 cells. CSML-0 cells weretransiently transfected with p21-luc along with p53 and mts1. Cells werecollected in 24 hours and relative luciferase activity was determined.

FIG. 25B depicts transactivation of p53-responsive reporters in Sao-2cells. Cells were transiently transfected with p21-luc along with p53and/or mts1. Cells were collected in 24 hours and relative luciferaseactivity was determined.

FIG. 26 depicts the effects of anti-Mts1 antibody administered to micebearing highly metastatic CSML-100 tumors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a new method for diagnosing metastaticcancer and for distinguishing metastatic tumors from benign tumors. Inparticular, the present invention demonstrates a heretofore unknownproperty of a mammalian gene, called mts-1, whose expression is about 10to about 100 fold higher in metastatic tumor cells, for example, of thelung, liver, kidney, mammary gland, epithelial, thyroid, leukemic,pancreatic, endometrial, ovarian, cervical, skin, colon or lymphoidtissue than in benign tumor cells or the corresponding normal cells.According to the present invention metastatic cancer of these and othertissues can be detected in patient's serum by a simple immunoassay.Moreover, metastatic cancer can also be diagnosed in tissue biopsies bythe present immunoassays or by in situ hybridization assays.

Metastasis is the formation of secondary tumors by cells derived from aprimary tumor. The metastatic process involves mobilization andmigration of primary tumor cells from the site of the primary tumor intonew tissues where the primary tumor cells induce the formation ofsecondary (metastatic) tumors. In accordance with the present inventivediscovery, the increased expression of the mts-1 gene in a cell ortissue is strongly indicative of metastatic potential. The presentinvention utilizes this unexpected and surprising correlation of highmammalian mts-1 gene expression with high metastatic potential to detector diagnose malignant cancer. Both the mammalian mts-1 nucleic acid andantibodies directed against mammalian mts-1 proteins are contemplatedfor use in the diagnosis of malignant cancer. The human mts-1 gene,depicted by one of the nucleotide sequences below, has been isolated forthe first time in the present invention.

ATG-GCG-TGC-CCT-CTG-GAG-AAG-GCC-CTG-GAT'GTG-ATG-GTG-TCC- SEQ ID NO:1ACC-TTC-CAC-AAG-TAC-TCG-GGC-AAA-GAG-GGT-GAC-AAG-TTC-AAG-CTC-AAC-AAG-TCA-GAG-CTA-AAG-GAG-CTG-CTG-ACC-CGG-GAG-CTG-CCC-AGC-TTC-TTG-GGG-AAA-AGG-ACA-GAT-GAA-GCT-GCT-TTC-CAG-AAG-CTG-ATG-AGC-AAC-TTG-GAC-AGC-AAC-AGG-GAC-AAC-GAG-GTG-GAC-TTC-CAA-GAG-TAC-TGT-GTC-TTC-CTG-TCC-TGC-ATC-GCC-ATG-ATG-TGT-AAC-GAA-TTC-TTT-GAA-GGC-TTC-CCA-GAT-AAG-CAG-CCC- AGG-AAG-AAA; orGGC-AGT-TGA-GGC-AGG-AGA-CAT-CAA-GAG-AGT-ATT-TGT-GCC- SEQ ID NO:3CTC-CTC-GGG-TTT-TAC-CTT-CCA-GCC-GAG-ATT-CTT-CCC-CTC-TCT-ACA-ACC-CTC-TCT-CCT-CAG-CGC-TTC-TTC-TTT-CTT-GGT-TTG-ATC-CTG-ACT-GCT-GTC-ATG-GCG-TGC-CCT-CTG-GAG-AAG-GCC-CTG-GAT-GTG-ATG-GTG-TCC-ACC-TTC-CAC-AAG-TAC-TCG-GGC-AAA-GAG-GGT-GAC-AAG-TTC-AAG-CTC-AAC-AAG-TCA-GAA-CTA-AAG-GAG-CTG-CTG-ACC-CGG-GAG-CTG-CCC-AGC-TTC-TTG-GGG-AAA-AGG-ACA-GAT-GAA-GCT-GCT-TTC-CAG-AAG-CTG-ATG-AGC-AAC-TTG-GAC-AGC-AAC-AGG-GAC-AAC-GAG-GTG-GAC-TTC-CAA-GAG-TAC-TGT-GTC-TTC-CTG-TCC-TGC-ATC-GCC-ATG-ATG-TGT-AAC-GAA-TTC-TTT-GAA-GGC-TTC-CCA-GAT-AAG-CAG-CCC-AGG-AAG-AAA-TGA-AAA-CTC-CTC-TGA-TGT-GGT-TGG-GGG-GTC-TGC-CAG-CTG-GGG-CCC-TCC-CTG-TCG-CCA-GTG-GGC-ACT-TTT-TTT-TTT-CCA-CCC-TGG-CTC-CTT-CAG-ACA-CGT-GCT-TGA-TGC-TGA-GCA-AGT-TCA-ATA-AAG-ATT-CTT-GGA-AGT-TTA,

wherein SEQ ID NO:3 is different from SEQ ID NO:1 at the underlinedpositions.

The amino acid sequence of the human mts-1 protein is depicted below(SEQ ID NO:2):

Met-Ala-Cys-Pro-Leu-Glu-Lys-Ala-Leu-Asp-Val-Met-Val-Ser-Thr-Phe-His-Lys-Tyr-Ser-Gly-Lys-Glu-Gly-Asp-Lys-Phe-Lys-Leu-Asn-Lys-Ser-Glu-Leu-Lys-Glu-Leu-Leu-Thr-Arg-Glu-Leu-Pro-Ser-Phe-Leu-Gly-Lys-Arg-Thr-Asp-q;lu-Ala-Ala-Phe-Gln-Lys-Leu-Met-Ser-Asn-Leu-Asp-Ser-Asn-Arg-Asp-Asn-Glu-Val-Asp-Phe-Gln-Glu-Tyr-Cys-Val-Phe-Led-Ser-Cys-Ile-Ala-Met-Met-Cys-Asn-Glu-Phe-Phe-Glu-Gly-Phe-Pro-Asp-Lys-Gln-Pro- Arg-Lys-Lys.

Other mammalian mts-1 genes are also contemplated.

The present invention also relates to a useful animal model system ofmetastasis for screening potential antimetastatic drugs and fordeveloping therapeutic regimens for cancer treatment. This model systemincludes non-metastasizing and metastasizing tumors that are maintainedby sequential transplantation from one mouse or rat to another, as wellas cultured cell lines, derived from these tumors, which retain themetastatic or non-metastatic potential of their parental tumors. Hence,these tumors or cell lines may be transplanted or injected into mice orrats to generate benign or metastatic tumors. Concurrently, drugs orother therapies with anti-tumorigenic or anti-metastatic potential, maybe introduced into the animal to test whether the formation of themetastatic and benign tumors is suppressed. This model system has highutility because of the predictable metastatic potential of the tumorsand cell lines therein and also because cell lines of differingmetastatic potential were derived from the same parental tumor and hencehave a common genetic and phenotypic make-up, except for theirmetastatic potential. Hence the animal model system of the currentinvention is highly controlled and has predictable metastatic potential.

The human mts-1 gene of the present invention was obtained by use ofmouse and rat mts-1 clones previously obtained by the present inventors.The mouse and rat mts-1 genes were obtained from cDNA libraries madefrom metastatic mouse and rat tumor RNAs. The mouse mts-1 gene has beenobtained from a highly metastatic cell line derived from a spontaneousmouse mammary carcinoma (CSML-100), while the mts-1 rat gene utilized inthe present invention was from a highly metastatic thyroid carcinoma,IR-6. Both the mouse and rat mts-1 genes were obtained by differentialhybridization of the respective cDNA libraries with a probe made from apool of mRNAs from highly metastatic tissues, and a probe made from apool of mRNAs from low metastatic tissues.

The human mts-1 gene was obtained from a cDNA library made by thepresent inventors from mRNA purified from cultured HeLa cells and fromcultured melanoma Wm64 cells. Clones hybridizing strongly to a mousemts-1 cDNA probe can be identified as being the human mts-1 homologue byDNA sequencing. Alternatively, a cDNA can be obtained by reversetranscription and polymerase chain reaction using mRNA purified frommetastatic cells, e.g. as provided in Miller 1988 Ann. Rev. Microbiol.42: 177.

There is a difference of seven amino acids between the mouse and humanmts-1 proteins, demonstrating that while the mouse and human proteinsare functionally related they are not identical structurally.

In another embodiment, the mouse, rat, and, in particular, the humanmts-1 genes of the present invention have been subcloned into convenientreplicable vectors for production of large amounts of mts-1 DNA andlarge amounts of sense or antisense mts-1 RNA. Convenient replicablevectors comprise the gene or a DNA fragment thereof of the presentinvention, an origin of replication which is operable in thecontemplated host, and, preferably, a selectable marker, for example, anantibiotic resistance marker. Many of these vectors are based on pBR322.Convenient replicable vectors which allow synthesis of RNA from the DNAof interest include Bluescript.™. (commercially available fromStratagene), pTrcHisB (Invitrogen) and others that are well known in theart.

The present invention also contemplates replicable expression vectorsallowing a higher level of expression of the mammalian mts-1 protein.Replicable expression vectors as described herein are generally DNAmolecules engineered for controlled expression of a desired gene,especially high level expression where it is desirable to produce largequantities of a particular gene product, or polypeptide. The vectorsencode promoters and other sequences to control expression of that gene,the gene being expressed, and an origin of replication which is operablein the contemplated host. Preferably the vector also encodes aselectable marker, for example, antibiotic resistance. Replicableexpression vectors can be plasmids, bacteriophages, cosmids and viruses.Any expression vector comprising RNA is also contemplated.

Preferred vectors of the present invention are derived from eukaryoticsources. Expression vectors that function in tissue culture cells areespecially useful, but yeast vectors are also contemplated. Thesevectors include yeast plasmids and minichromosomes, retrovirus vectors,BPV (bovine papilloma virus) vectors, baculovirus vectors, SV40 basedvectors and other viral vectors. SV40-based vectors and retrovirusvectors (e.g., murine leukemia viral vectors) are preferred. Tissueculture cells that are used with eukaryotic replicable expressionvectors include Sf21 cells, CV-1 cells, COS-1 cells, NIH3T3 cells, mouseL cells, HeLa cells and such other cultured cell lines known to oneskilled in the art.

A baculovirus expression system can be used to produce large amounts ofmts-1 polypeptides in cultured insect cells. The post-translationalprocessing of polypeptides produced in such insect cells is similar tothat of mammalian cells. Production of polypeptides in insects istherefore advantageous, particularly when one seeks to mimic the exactfunction or antigenic properties of the natural polypeptide. Moreover,mts-1 polypeptides expressed in the baculovirus system are producedwithout the need for a fused heterologous polypeptide because the mts-1start codon is used as the translational start site.

Methods for producing polypeptides in the baculovirus expression systemare known to the skilled artisan. See for example Miller 1988 Ann. Rev.Microbiol. 42: 177. In general, a modified Autographa californicanuclear polyhedrosis virus propagated in Sf21 cells is used forpolypeptide expression. This modified virus is produced bycotransfection of a small transfer vector, encoding an mts-1polypeptide, with a viral expression vector which has been linearizedwithin an essential gene.

Once inside the cell, the linearized expression vector can undergorecombination with the transfer vector or simply recircularize. However,only recombination gives rise to viable viruses because the function ofthe essential gene is lost by recircularization. Recombinant expressionviruses are detected by formation of plaques. The present invention alsocontemplates prokaryotic vectors that may be suitable for expression ofthe mammalian mts-1 gene, including bacterial and bacteriophage vectorsthat can transform such hosts as E. coli, B. subtilis, Streptomyces sps.and other microorganisms. Many of these vectors are based on pBR322including Bluescript.™. (commercially available from Stratagene) and arewell known in the art. Bacteriophage vectors that are used in theinvention include lambda and M13.

Sequence elements capable of effecting expression of the human mts-1gene include promoters, enhancer elements, transcription terminationsignals and polyadenylation sites. Promoters are DNA sequence elementsfor controlling gene expression, in particular, they specifytranscription initiation sites. Prokaryotic promoters that are usefulinclude the lac promoter, the trp promoter, and P_(L) and P_(R)promoters of lambda and the T7 polymerase promoter. Eukaryotic promotersare especially useful in the invention and include promoters of viralorigin, such as the SV40 late promoter and the Moloney Leukemia VirusLTR, Murine Sarcoma Virus (MSV) LTR, yeast promoters and any promotersor variations of promoters designed to control gene expression,including genetically-engineered promoters. Control of gene expressionincludes the ability to regulate a gene both positively and negatively(i.e., turning gene expression on or off) to obtain the desired level ofexpression.

The replicable expression vectors of the present invention can be madeby ligating part or all of the mts-1 coding region in the sense orantisense orientation to the promoter and other sequence elements beingused to control gene expression. This juxtapositioning of promoter andother sequence elements with the mts-1 gene allows the production oflarge amounts of sense or antisense mts-1 mRNA. Large amounts of themts-1 protein can also be produced which are useful not only foranti-mts-1 antibody production but also for analysis of the function ofmts-1 in metastatic cancer as well as for designing therapies formetastatic cancer.

As one example of an appropriate expression vector for the human mts-1gene, the present invention provides the pEMSVscribe2 vector whichexpresses the human mts-1 gene of this invention.

In another example, large quantities of the mts-1 specific protein wereexpressed in an E. coli host using the inducible bacterial vectorpTrcHisB (FIG. 10a). Murine mts-1 cDNA was subcloned in frame into aBamHI-KpnI site with the multiple cloning site of pTrcHisB to generateplasmid pTBM1. The fusion protein expressed by pTBM1 has 6 tandemhistidine residues which allow easy purification of the fusion proteinbecause of the high affinity of such tandem histidines for a Ni.sup.++charged resin. The fusion protein also has an enterokinase specificcleavage site permitting removal of the histidines from the mts-1protein product. Expression of the mts-1 fusion protein encoded by pTBM1can be induced by IPTG. Similar human mts-1 cDNA constructs have alsobeen generated.

Therefore, one skilled in the art has available many choices ofreplicable expression vectors, compatible hosts and well-known methodsfor making and using the vectors. Recombinant DNA methods are found inany of the myriad of standard laboratory manuals on genetic engineering.

The present invention is also directed to the detection of metastaticcancer in tissue specimens by use of the mts-1 DNA as a nucleic acidprobe for detection of mts-1 mRNA, or by use of antibodies directedagainst the mts-1 protein.

The nucleic acid probe of the present invention may be any portion orregion of a mammalian mts-1 RNA or DNA sufficient to give a detectablesignal when hybridized to mts-1 mRNA derived from a tissue sample. Thenucleic acid probe produces a detectable signal because it is labeled insome way, for example because the probe was made by incorporation ofnucleotides linked to a “reporter molecule”.

A “reporter molecule”, as used in the present specification and claims,is a molecule which, by its chemical nature, provides an analyticallyidentifiable signal allowing detection of the hybridized probe.Detection may be either quantitative or quantitative. The most commonlyused reporter molecules in this type of assay are either enzymes,fluorophores or radionuclides covalently linked to nucleotides which areincorporated into a mts-1 DNA or RNA. Commonly used enzymes includehorseradish peroxidase, alkaline phosphatase, glucose oxidase andβ-galactosidase, among others. The substrates to be used with thespecific enzymes are generally chosen for the production, uponhydrolysis by the corresponding enzyme, of a detectable color change.For example, p-nitrophenyl phosphate is suitable for use with alkalinephosphatase conjugates; for horseradish peroxidase,1,2-phenylenediamine, 5-aminosalicyclic acid or tolidine are commonlyused.

Incorporation into a mts-1 DNA probe may be by nick translation, randomoligo priming, by 3′ or 5′ end labeling, by labeled single-stranded DNAprobes using single-stranded bacteriophage vectors (e.g. M13 and relatedphage), or by other means, (Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual. Cold Spring Harbor Laboratory Press. Pages10.1-10.70). Incorporation of a reporter molecule into a mts-1 RNA probemay be by synthesis of mts-1 RNA using T3, T7, Sp6 or other RNApolymerases (Sambrook et al., supra: 10.27-10.37).

Detection or diagnosis of metastatic cancer by the nucleic acid probe ofthe present invention can be by a variety of hybridization techniqueswhich are well known in the art. In one embodiment, patient tissuespecimens are sectioned and placed onto a standard microscope slide,then fixed with an appropriate fixative. The mts-1 RNA or DNA probe,labeled by one of the techniques described above, is added. The slide isthen incubated at a suitable hybridization temperature (generally 37° C.to 55° C.) for 1-20 hours. Non-hybridized RNA or DNA probe is thenremoved by extensive, gentle washing. If a non-radioactive reportermolecule is employed in the probe, the suitable substrate is applied andthe slide incubated at an appropriate temperature for a time appropriateto allow a detectable color signal to appear as the slide is visualizedunder light microscopy. Alternatively, if the mts-1 probe is labeledradioactively, slides may be dipped in photoemulsion after hybridizationand washing, and the signal detected under light microscopy afterseveral days, as exposed silver grains.

Metastatic cancer can also be detected from RNA derived from tissuespecimens by the mts-1 nucleic acid probe. RNA from specimens can befixed onto nitrocellulose or nylon filters, and well-known filterhybridization techniques may be employed for detection of mts-1 geneexpression.

Specimen mRNA can be purified, or specimen cells may be simply lyzed andcellular mRNA fixed unto a filter. Specimen mRNA can be sizefractionated through a gel before fixation onto a filter, or simply dotblotted unto a filter.

In another embodiment, the mts-1 nucleic acid detection system of thepresent invention also relates to a kit for the detection of mts-1 mRNA.In general, a kit for detection of mts-1 mRNA contains at least onemts-1 nucleic acid. Such an mts-1 nucleic acid can be a probe having anattached reporter molecule or the mts-1 nucleic acid can be unlabelled.The unlabelled mts-1 nucleic acid can be modified by the kit user toinclude a reporter molecule or can act as a substrate for producing alabelled mts-1 probe, for example by nick translation or RNAtranscription.

In another embodiment, the kit is compartmentalized: a first containercan contain mts-1 RNA at a known concentration to act as a standard orpositive control, a second container can contain mts-1 DNA suitable forsynthesis of a detectable nucleic acid probe, and a third and a fourthcontainer can contain reagents and enzymes suitable for preparing saidmts-1 detectable probe. If the detectable nucleic acid probe is made byincorporation of an enzyme reporter molecule, a fifth or sixth containercan contain a substrate, or substrates, for the enzyme provided.

In accordance with the present invention, the mts-1 protein or portionsthereof can be used to generate antibodies useful for the detection ofthe mts-1 protein in clinical specimens. Such antibodies may bemonoclonal or polyclonal. Additionally, it is within the scope of thisinvention to include second antibodies (monoclonal or polyclonal)directed to the anti-mts-1 antibodies. The present invention furthercontemplates use of these antibodies in a detection assay (immunoassay)for the mts-1 gene product.

The present invention further contemplates antibodies directed againstthe mammalian, including rat, mouse and human, mts-1 proteins orpolypeptides. These antibodies may be generated by using the entiremts-1 protein as an antigen or by using short peptides, encodingportions of the mts-1 protein, as antigens. When peptides arecontemplated they have at least about 4 amino acids and preferably atleast about 10 amino acids.

Preferably, specific peptides encoding unique portions of the mammalianmts-1 gene are synthesized for use as antigens for obtaining mts-1antibodies. This is done because mts-1 encodes a calcium binding domainwhose sequence, and hence antigenicity, is similar to other calciumbinding proteins. By utilizing peptides encoding sequences lying outsidethe calcium binding domain, cross-reactivity of the anti-mts-1antibodies towards other calcium binding proteins easily can be avoided.Accordingly, peptide sequences are tested for sequence homologies bysearching protein sequence data banks before peptides are actuallysynthesized. Among the various mts-1 peptides that can be used, fourpeptides encoding a portion of the human mts-1 sequence shown below,have already been used to generate antibodies:

1) Unique peptide encoding amino acids 2-11 of the mts-1 protein (SEQ IDNO:4): Ala-Cys-Pro-Leu-Glu-Lys-Ala-Leu-Asp-Val;

2) Peptide encoding the calcium binding domain of the mts-1 protein(amino acids 22-37, SEQ ID NO:5):Lys-Glu-Gly-Asp-Lys-Phe-Lys-Leu-Asn-Lys-Ser-Glu-Leu-Lys-Glu-Leu;

3) Unique peptide encoding amino acids 42-54 of the mts-1 protein (SEQID NO:6): Leu-Pro-Ser-Phe-Leu-Gly-Lys-Arg-Thr-Asp-Glu-Ala-Ala;

4) Unique peptide encoding amino acids 87-101 of mts-1 protein (SEQ IDNO:7): Asn-Glu-Phe-Phe-Glu-Gly-Phe-Pro-Asp-Lys-Gln-Pro-Arg-Lys-Lys.

Polyclonal antibodies directed against the mts-1 protein are prepared byinjection of a suitable laboratory animal with an effective amount ofthe peptide or antigenic component, collecting serum from the animal,and isolating specific sera by any of the known immunoadsorbenttechniques. Animals which can readily be used for producing polyclonalanti-mts-1 antibodies include chickens, mice, rabbits, rats, goats,horses and the like. Chickens are preferred because a better immuneresponse can be obtained and because antibodies can be collected fromeggs rather than by bleeding. Although the polyclonal antibodiesproduced by this method are utilizable in virtually any type ofimmunoassay, they are generally less favored because Of the potentialheterogeneity of the product.

The use of monoclonal antibodies in the diagnostic or detection assaysof the present invention is particularly preferred because largequantities of antibodies, all of similar reactivity, may be produced.The preparation Of hybridoma cell lines for monoclonal antibodyproduction is done by fusing an immortal cell line and the antibodyproducing lymphocytes. This can be done by techniques which are wellknown to those who are skilled in the art. (See, for example, Harlow, E.and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Press,1988; or Douillard, J. Y. and Hoffman, T., “Basic Facts AboutHybridomas”, in Compendium of Immunology Vol. II, L. Schwartz (Ed.),1981.

Unlike the preparation of polyclonal sera, the choice of animal formonoclonal antibody preparation is dependent on the availability ofappropriate immortal cell lines capable of fusing with the monoclonalantibody producing lymphocytes derived from the immunized animal. Mouseand rat have been the animals of choice for hybridoma technology and arepreferably used. Humans can also be utilized as sources for antibodyproducing lymphocytes if appropriate immortalized human (or nonhuman)cell lines are available. For the purpose of making the monoclonalantibodies of the present invention, the animal of choice may beinjected with, from about 0.01 mg to about 20 mg of the purified mts-1antigen. Usually the injecting material is emulsified in Freund'scomplete adjuvant. Boosting injections are generally also required. Theseparate immortalized cell lines obtained by cell fusion may be testedfor antibody production by testing the cell culture media for theability to find the appropriate antigen.

Lymphocytes can be obtained by removing the spleen or lymph nodes ofimmunized animals in a sterile fashion. Alternately, lymphocytes can bestimulated or immunized in vitro, as described, for example, in C.Reading J. Immunol. Meth. 53:261-291 1982. To immortalize the monoclonalantibody producing lymphocytes, the lymphocytes must be fused toimmortalized cells. A number of cell lines suitable for fusion have beendeveloped, and the choice of any particular line for hybridizationprotocols is directed by any one of a number of criteria such as speed,uniformity of growth characteristics, deficiency of its metabolism for acomponent of the growth medium, and potential for good fusion frequency.Intraspecies hybrids, particularly between like strains, work betterthan interspecies fusions.

Several cell lines are available, including mutants selected for theloss of ability to create myeloma immunoglobulin. Included among theseare the following mouse myeloma lines: MPC₁₁ -X45-6TG, P3 NS1/1-Ag4-1,P3-X63-Ag14 (all BALB/C derived), Y3′Ag1.2.3 (rat), and U266 (human).

Cell fusion can be induced either by virus, such as Epstein-Barr orSendai virus, or polyethylene glycol. Polyethylene glycol (PEG) is themost efficacious agent for the fusion of mammalian somatic cells. PEGitself may be toxic for cells, and various concentrations should betested for effects on viability before attempting fusion. The molecularweight range of PEG may be varied from 1,000 to 6,000. It give bestresults when diluted to from about 20% to about 70% w/w in saline orserum-free medium. Exposure to PEG at 37° C. for about 30 seconds ispreferred in the present case, utilizing murine cells. Extremes oftemperature (i.e. about 45° C.) are avoided, and preincubation of eachcomponent of the fusion system at 37° C. prior to fusion gives optimumresults. The ratio between lymphocytes and immortalized cells optimizedto avoid cell fusion amongst lymphocytes ranges of from about 1:1 toabout 1:10.

The successfully fused cells can be separated from the immortalized cellline by any technique known by the art. The most common and preferredmethod is to choose an immortalized cell line which is HypoxanthineGuanine Phosphoribosyl Transferase (HGPRT) deficient. Since these cellswill not grow in an aminopterin-containing medium, only hybrids oflymphocytes and immortalized cells will grow. The aminopterin-containingmedium is generally composed of hypoxanthine 1×10⁻⁴ M, aminopterin 1×10⁵M, and thymidine 3×10⁻⁵ M, commonly known as the HAT medium. Fused cellsare generally grown for two weeks and then fed with either regularculture medium or hypoxanthine, thymidine-containing medium.

The fused cell colonies are then tested for the presence of antibodiesthat recognize the mts-1 protein. Detection of hybridoma antibodies canbe performed using an assay where the antigen is bound to a solidsupport and allowed to react to hybridoma supernatants containingputative antibodies. The presence of antibodies may be detected by“sandwich” techniques using a variety of indicators. Most of the commonmethods are sufficiently sensitive for use in the range of antibodyconcentrations secreted during hybrid growth.

Cloning of hybrid cells can be carried out after 20-25 days of cellgrowth in selected medium. Cloning can be performed by cell limitingdilution in fluid phase or by directly selecting single cells growing insemi-solid agarose. For limiting dilution, cell suspensions are dilutedserially to yield a statistical probability of having only one cell perwell. For the agarose techniques, hybrids are seeded in a semisolidupper layer, over a lower layer containing feeder cells. The coloniesfrom the upper layer may be picked up and eventually transferred towells.

Antibody-secreting hybrid cells can be grown in various tissue cultureflasks, yielding supernatants with variable concentrations ofantibodies. In order to obtain higher concentrations, hybrid cells maybe transferred into animals to obtain inflammatory ascites.Antibody-containing ascites can be harvested 8-12 days afterintraperitoneal injection. The ascites contain a higher concentration ofantibodies but include both monoclonals and immunoglobulins from theinflammatory ascites. Antibody purification may then be achieved by, forexample, affinity chromatography.

One embodiment of the present invention is directed to a method fordiagnosing metastatic cancer by contacting or applying an antibodyreactive with an mts-1 polypeptide to a tissue or blood sample takenfrom an individual to be tested for metastatic cancer. Formation of anantigen-antibody complex in this immunoassay is diagnostic of metastaticcancer.

In a preferred embodiment, the present invention provides a method fordiagnosing metastatic cancer which involves contacting serum from anindividual to be tested for such cancer with an antibody reactive with amammalian mts-1 protein or an antigenic fragment thereof, for a time andunder conditions sufficient to form an antigen-antibody complex, anddetecting the antigen-antibody complex.

The presence of the mts-1 protein, or its antigenic components, in apatient's serum, tissue or biopsy sample can be detected utilizingantibodies prepared as above, either monoclonal or polyclonal, invirtually any type of immunoassay. A wide range of immunoassaytechniques are available as can be seen by reference to Harlow, et al.(Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988) andU.S. Pat. Nos. 4,016,043 and 4,424,279. This, of course, includes bothsingle-site and two-site, or “sandwich” of the non-competitive types, aswell as in traditional competitive binding assays. Sandwich assays areamong the most useful and commonly used assays. A number of variationsof the sandwich assay technique exist, and all are intended to beencompassed by the present invention. Briefly, in a typical forwardassay, an unlabeled antibody is immobilized in a solid substrate and thesample to be tested brought into contact with the bound molecule. Aftera suitable period of incubation, for a period of time sufficient toallow formation of an antibody-antigen binary complex, a secondantibody, labeled with a reporter molecule capable of producing adetectable signal is then added and incubated, allowing tie sufficientfor the formation of a ternary complex of antibody-labeled antibody. Anyreacted material is washing way, and the presence of the antigen isdetermined by observation of a signal produced by the reporter molecule.The results may either be qualitative, by simple observation of thevisible signal, or may be quantitated by comparing with a control samplecontaining known amounts of hapten. Variations on the forward assayinclude a simultaneous assay, in which both sample and labeled antibodyare added simultaneously to the bound antibody, or a reverse assay inwhich the labeled antibody and sample to be tested are first combined,incubated and then added to the unlabeled surface bound antibody. Thesetechniques are well known to those skilled in the art, and then possiblyof minor variations will be readily apparent. As used herein, “sandwichassay” is intended to encompass all variations on the basic two-sitetechnique.

The mts-1 protein may also be detected by a competitive binding assay inwhich a limiting amount of antibody specific for the mts-1 protein iscombined with specified volumes of samples containing an unknown amountsof the mts-1 protein and a solution containing a detectably labeledknown amount of the mts-1 protein. Labeled and unlabeled molecules thencompete for the available binding sites on the antibody. Phaseseparation of the free and antibody-bound molecules allows measurementof the amount of label present in each phase, thus indicating the amountof antigen or hapten in the sample being tested. A number of variationsin this general competitive binding assays currently exist.

In any of the known immunoassays, for practical purposes, one of theantibodies or the antigen will be typically bound to a solid phase and asecond molecule, either the second antibody in a sandwich assay, or, ina competitive assay, the known amount of antigen, will bear a detectablelabel or reporter molecule in order to allow visual detection of anantibody-antigen reaction. When two antibodies are employed, as in thesandwich assay, it is only necessary that one of the antibodies bespecific for the mts-1 protein or its antigenic components. Thefollowing description will relate to a discussion of a typical forwardsandwich assay; however, the general techniques are to be understood asbeing applicable to any of the contemplated immunoassays.

In the typical forward sandwich assay, a first antibody havingspecificity for the mts-1 protein or its antigenic components is eithercovalently or passively bound to a solid surface. The solid surface istypically glass or a polymer, the most commonly used polymers beingcellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene. The solid supports may be in the form of tubes, beads,discs or microplates, or any other surface suitable for conducting animmunoassay. The binding processes are well-known in the art andgenerally consist of cross-linking covalently binding or physicallyadsorbing the molecule to the insoluble carrier. Following binding, thepolymer-antibody complex is washed in preparation for the test sample.An aliquot of the sample to be tested is then added to the solid phasecomplex and incubated at a suitable temperature ranging from about 4° C.to about 37° C. (for example 25° C.) for a period of time sufficient toallow binding of any subunit present in the antibody. The incubationperiod will vary but will generally be in the range of about 2-40minutes to several hours. Following the incubation period, the antibodysubunit solid phase is washed and dried and incubated with a secondantibody specific for a portion of a mts-1 hapten. The second antibodyis linked to a reporter molecule which is used to indicate the bindingof the second antibody to the hapten.

By “reporter molecule”, as used in the present specification and claims,is meant a molecule which, by its chemical nature, provides ananalytically identifiable signal which allows the detection ofantigen-bound antibody. Detection may be either qualitative orquantitative. The most commonly used reporter molecules in this type ofassay are either enzymes, fluorophores or radionuclide containingmolecules. In the case of an enzyme immunoassay, an enzyme is conjugatedto the second antibody, generally by means of glutaraldehyde orperiodate. As will be readily recognized, however, a wide variety ofdifferent conjugation techniques exist, which are readily available tothe skilled artisan. Commonly used enzymes include horseradishperoxidase, glucose oxidase, β-galactosidase and alkaline phosphates,among others. The substrates to be used with the specific enzymes aregenerally chosen for the production, upon hydrolysis by thecorresponding enzyme, of a detectable color change. For example,p-nitrophenyl phosphate is suitable for use with alkaline phosphataseconjugates; for peroxidase conjugates, 1,2-phenylenediamine,5-aminosali-cyclic acid, or tolidine are commonly used. It is alsopossible to employ fluorogenic substrates, which yield a fluorescentproduct rather than the chromogenic substrates noted above. In allcases, the enzyme-labeled antibody is added to the first antibody haptencomplex, allowed to bind, and then the excess reagent is washed away. Asolution containing the appropriate substrate is then added to theternary complex of antibody-antigen-antibody. The substrate will reactwith the enzyme linked to the second antibody, giving a qualitativevisual signal, which may be further quantitated, usuallyspectrophotometrically, to give an indication of the amount of haptenwhich was present in the sample.

Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody absorbs the light energy,inducing a state of excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. The fluorescent labeled antibody is allowed to bind to thefirst antibody-hapten complex. After washing off the unbound reagent,the remaining ternary complex is then exposed to the light of theappropriate wavelength, the fluorescence observed indicates the presenceof the hapten of interest. Immunofluorescence techniques are very wellestablished in the art. However, other reporter molecules, such asradioisotope, chemiluminescent or bioluminescent molecules, may also beemployed. It will be readily apparent to the skilled technician how tovary the procedure to suit the required purpose.

In another embodiment, the antibodies directed against the mts-1 proteinmay be incorporated into a kit for the detection of the mts-1 protein.Such a kit may encompass any of the detection systems contemplated anddescribed herein, and may employ either polyclonal or monoclonalantibodies directed against the mts-1 protein. Both mts-1 antibodiescomplexed to a solid surface described above or soluble mts-1 antibodiesare contemplated for use in a detection kit. A kit of the presentinvention has at least one container having an antibody reactive with amammalian mts-1 polypeptide. However, the present kits can have othercomponents. For example, the kit can be compartmentalized: the firstcontainer contains mts-1 protein as a solution, or bound to a solidsurface, to act as a standard or positive control, the second containercontains anti-mts-1 primary antibodies either free in solution or boundto a solid surface, a third container contains a solution of secondaryantibodies covalently bound to a reporter molecule which are reactiveagainst either the primary antibodies or against a portion of the mts-1protein not reactive with the primary antibody. A fourth and fifthcontainer contains a substrate, or reagent, appropriate forvisualization of the reporter molecule.

The subject invention therefore encompasses polyclonal and monoclonalantibodies useful for the detection of mts-1 protein as a means ofdiagnosing metastatic cancer. Said antibodies may be prepared asdescribed above, then purified, and the detection systems contemplatedand described herein employed to implement the subject invention.

The present invention also contemplates treating metastatic cancers andtumors by inactivating, destroying or nullifying the mts-1 gene orprotein, or cells expressing the mts-1 gene. The treatment of cancer, asdescribed in the specification and claims, contemplates preferably lung,liver, kidney, thyroid, mammary gland, leukemic, pancreatic,endometrial, ovarian, cervical, skin, colon or lymphoid cancers. Forexample, the antibodies, prepared as described above, may be utilized toinactivate mts-1 protein expressing cells: either unconjugatedanti-mts-1 antibodies or anti-mts-1 antibodies conjugated to a toxin maybe employed in the therapy of cancer.

Moreover, the present invention provides a method of inhibitingmetastasis in a cancerous cell by providing to the cancerous cell anucleic acid encoding an antisense mts-1 nucleotide sequence. Forexample, such an antisense nucleic acid can have at least 10 nucleotidesof the antisense strand of SEQ ID NO:1 or SEQ ID NO:3. Preferably, theantisense mts-1 nucleic acids of the present invention have at least 15or 17 nucleotides.

In one embodiment, this method employs an expression vector including anucleic acid encoding an antisense nucleotide sequence for mts-1operably linked to a segment of the vector which can effect expressionof an antisense mts-1 RNA. Any of the foregoing expression vectors whichcan express high levels of mts-1 RNA can be used for this methodincluding, e.g., pTrcHis.

According to the present invention, antisense mts-1 nucleic acids caninhibit metastatic cancer by binding to sense mts-1 mRNA. Such bindingcan either prevent translation of mts-1 protein or destroy mts-1 sensemRNA, e.g., through the action of RNaseH. Accordingly, less mts-1protein is available to potential metastatic tumor cells and metastasisof these cells is prevented.

Another embodiment of the present invention contemplates pharmaceuticalcompositions containing, for example, an antibody reactive with amammalian mts-1 polypeptide, an antisense mts-1 nucleic acid or themts-1 protein. The mts-1 protein is known to bind calcium and has a rolein the growth of cells (Linzer, et al., Proc. Natl. Acad. Sci. USA80:4271-4275, 1983; Jackson-Grusby, et al., Nuc. Acids. Res.15:6677-6689; Goto et al., J. Biochem. 103:48-53, 1988). The mts-1protein is also very closely related to 42A, a gene thought to have arole in nerve cell growth (Masiakowski, et al. Proc. Natl. Acad. Sci.USA 85:1277-1281, 1988). The mts-1 protein may also have a role in thedifferentiation of myoepithelial cells (Barraclough, et al., J. Mol.Biol. 198:13-20, 1987). Hence the human mts-1 protein may be clinicallyuseful, for example, in stimulating cells in general or preferably,nerve cells, to grow, and further, in promoting the differentiation ofmyoepithelial cells.

The active ingredients of a pharmaceutical composition containing themts-1 protein or anti-mts-1 antibodies and antisense mts-1 nucleic acids(i.e. anti-cancer reagents) are contemplated to exhibit effectivetherapeutic activity, for example, in promoting cell growth, or fortreating cancer, respectively. Thus the active ingredients of thetherapeutic compositions containing mts-1 protein cell proliferativeactivity or anti-cancer reagents, are administered in therapeuticamounts which depend on the particular disease. For example, from about0.5 μg to about 2000 mg per kilogram of body weight per day may beadministered. The dosage regimen may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. A decidedpractical advantage is that the active compound may be administered in aconvenient manner such as by the oral, intravenous (where watersoluble), intramuscular, subcutaneous, intranasal, intradermal orsuppository routes. Depending on the route of administration, the activeingredients which comprise mts-1 proteins or anti-cancer reagents may berequired to be coated in a material to protect said ingredients from theaction of enzymes, acids and other natural conditions which mayinactivate said ingredients. For example, the low lipophilicity of mts-1protein, and some anti-cancer reagents, may allow them to be destroyedin the gastrointestinal tract by enzymes capable of cleaving peptide ornucleotide bonds and in the stomach by acid hydrolysis. In order toadminister mts-1 protein or anti-cancer reagents by other thanparenteral administration, they should be coated by, or administeredwith, a material to prevent its inactivation. For example, mts-1 proteinor anti-cancer reagents may be administered in an adjuvant,co-administered with enzyme inhibitors or in liposomes. Adjuvantscontemplated herein include resorcinols, non-ionic surfactants such aspolyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzymeinhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DFP) and trasylol. Liposomes includewater-in-oil-in-water P40 emulsions as well as conventional liposomes.

The active compounds may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof, and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases the form must be sterile and mustbe fluid to the extend that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, liquid polyethylene glycol, and the like), suitable mixturesthereof and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. The preventions of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When the mts-1 protein or anti-cancer reagents are suitably protected asdescribed above, the active compound may be orally administered, forexample, with an inert diluent or with an assimilable edible carrier, orit may be enclosed in hard or soft shell gelatin capsule, or it may becompressed into tablets, or it may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompound may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Compositions or preparationsaccording to the present invention are prepared so that an oral dosageunit form contains between about 0.5 μg and 2000 μg of active compound.

The tablets, troches, pills, capsules, and the like, as described above,may also contain the following: a binder such as gum gragacanth, acacia,corn starch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acid,and the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavoringagent such as peppermint, oil or wintergreen or cherry flavoring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release preparations and formulations.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of the active materialcalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The specification for the noveldosage unit forms of the invention are dictated by and directlydepending on (a) the unique characteristics of the active material andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such as active materialfor the treatment of disease in living subjects having a diseasedcondition in which bodily health is impaired as herein disclosed indetail.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from 0.5 μg to about 2000 μg.Expressed in proportions, the active compound is generally present infrom about 10 .mu.g to about 2000 mg/ml of carrier. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and adsorption delaying agents, and the like. The useof such media gents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The present invention is further directed to treating tumors byinterefering with, destroying or nullifying the function of mts-1 geneor protein.

In accordance with the present invention, Mts-1 interferes with thefunction of tumor suppressor p53 by binding to p53. The presentinventors have demonstrated that binding with Mts-1 inhibits p53phosphorylation by PKC, represses the DNA-binding activity of p53 andreduces the transactivation capacity of p53. The present invention hasfurther identified that the Mts-1 protein binds to the C-terminal regionof p53. Accordingly, the present invention provides compositions andmethods useful for treating tumors by interfering with the expression orfunction of Mts-1, e.g., intercepting the interaction between Mts-1 andtumor suppressor p53.

The present invention provides peptides which prevent p53 from bindingto Mts1, e.g., a peptide comprising aa 289-393 of human p53, a peptidecomprising aa 360-393 of human p53, a peptide comprising aa 289-390 ofmurine p53, a peptide comprising aa 360-390 of murine p53, a peptidecomprising the C-terminal nonmuscle myosin heavy chain, a peptidecomprising amino acid 1909-1937 of human nonmuscle myosin heavy chain.Functional fragments and analogs of these peptides are also contemplatedby the present invention.

“Functional fragments” refer to peptide fragments that have the samefunction as the peptide in issue, namely, the function of interferingthe Mts1-p53 interaction by binding to Mts-1.

By “analogs” it means variants of a peptide in issue. The variationsinclude substitutions, insertions or deletions of one or more amino acidresidues, or modifications of the side chains of the amino acidresidues. Thus, analogs of a peptide can include homologous peptidesfrom other mammalian species, peptides containing non-natural amino acidresidues, peptides having chemical modifications on the side groups ofamino acid residues, as well as peptides artificially designed toresemble the three dimensional structure of the binding site on humanp53.

A variety of techniques are available to those skilled in the art tomake various fragments or analogs of p53. Such techniques includestandard chemical synthesis, recombinant expression, and structuralmodeling (also called ‘mimetics’). The sequences of p53 from a number ofmammalian species are highly conserved and are available to thoseskilled in the art, e.g., via Databases such as GenBank.

Mimetics is a well-known technique involving the design of compoundswhich contain functional groups arranged in a manner mimicking that ofthe original, lead compound. Mimetics is desirable where the synthesisof the original compound is difficult, or where the original compound isunsuitable for a particular method of administration (e.g., orally) assuch compound may tend to be degraded too quickly by proteases in thealimentary canal. Mimetic design, synthesis and testing can avoidlaborious screenings of a large number of molecules for a targetproperty.

A variety of methods can be employed to determine whether a fragment oranalog is “functional”, i.e., capable of interfering the interactionbetween Mts-1 and p53 by binding to Mts-1. In one format, for example, asample containing Mts-1 proteins can be contacted with a test compoundfor a period of time sufficient to allow binding of the test compound toMts-1. Then, the sample is contacted with p53, and the amount ofcomplexes formed between Mts-1 and p53 can be measured and compared tothe amount of complexes formed in the absence of the test compound. Adecrease in the value determines the test compound as a compound whichprevents the binding of Mts-1 to p53.

An alternative format to carry out the method of the present inventioncan include as a first step, contacting a sample containing Mts-1 withp53 for a time sufficient to allow p53 to bind to Mts-1 and measuringthe amount of complexes formed between Mts-1 and p53 in the sample.Afterwards, the sample is contacted with a test compound for anappropriate period of time. Compounds which displace p53 from the priorformed p53-Mts1 complexes can be identified as a compound which preventsthe binding of Mts-1 to p53.

A variety of biochemical assays and immunoassays can be employed formeasuring the amount of the Mts1-p53 complexes formed in a sample. Theproteins can be conveniently labeled with, e.g., isotope, biotinylgroup, or fluorescein, to facilitate the detection. The assays can becarried out in a variety of formats, such as immunoprecipitation, Farwestern blot analysis, RIA, ELISA, forward or reverse sandwich assays,peptide competition binding assays, and the like. Peptides identified byusing any of the above-described biochemical assays or immunoassays canbe further tested and confirmed in functional assays, such asDNA-binding gel shift assay, or a reporter expression assay, which arefurther described in the Examples hereinafter.

The present invention also contemplates pharmaceutical compositionswhich include, as an active ingredient, an Mts1-p53 binding interceptingpeptide as described hereinabove, and a pharmaceutically acceptablecarrier.

In another aspect of the present invention, Mts1-p53binding-intercepting peptides or nucleic acid molecules encoding thereofare used for treating tumors.

By “treating a tumor” it means that the tumor growth or metastasis issignificantly inhibited, as indicated by reduced tumor volumn or reducedoccurrences of tumor metastasis. Tumor growth can be determined, e.g.,by examining the tumor volume via routine procedures (such as obtainingtwo-dimensional measurements with a dial caliper). Tumor metastasis canbe determined by examining the appearance of tumor cells in secondarysites or examining the metastatic potential of biopsied tumor cells invitro using various laboratory procedures.

According to the present invention, the tumors which can be treated byusing the methods of the present invention may include, but are notlimited to, melanoma, lymphoma, plasmocytoma, sarcoma, glioma, thymoma,leukemia, breast cancer, prostate cancer, colon cancer, esophagealcancer, brain cancer, lung cancer, ovary cancer, cervical cancer,hepatoma, and other neoplasms known in the art.

The present invention contemplates particularly p53-related tumors. Theterm “p53-related” refers to tumor cells in which wild-type (wt) p53 isabsent, disabled or otherwise mutated.

A variety of methods are available to those skilled in the art fordetermining whether a tumor is “p53-related”. For example, EPA 518 650(Vogelstein, B. et al.) describes a method for detecting p53 in cellularextracts using DNA sequences that are specific for p53 binding. WO94/11533 describes determining the presence of functional p53 in cellsby measuring mRNA or protein encoded by a growth-arrest and DNA-damageinducible gene, GADD45. U.S. Pat. No. 5,876,711 describes a rapid invivo method for determining the status of tumor suppressor proteins inpatient tumor cells. Such method includes contacting the tumor cellswith a first and second polynucleotide sequence such that they are takenup by the tumor cells. The first polynucleotide sequence encodes areporter molecule that is operably linked to the second polynucleotidesequence which sequence binds the tumor suppressor. Binding of the tumorsuppressor causes the expression of the reporter molecule, which is thendetected or quantitated.

In one embodiment, the present invention provides methods of treating atumor in a subject by administering to the subject, a therapeuticallyeffective amount of a peptide which prevents the binding of Mts-1 top53. Preferred binding intercepting peptides have been describedhereinabove.

In another embodiment, the present invention provides methods oftreating a tumor in a subject by administering to the subject, atherapeutically effective amount of a nucleic acid molecule coding for apeptide which prevents the binding of Mts-1 to p53.

Preferably, such nucleotide sequence is provided in an expressionvector. Preferred expression vectors for use in a therapeuticcomposition include any appropriate gene therapy vectors, such asnonviral (e.g., plasmid vectors), retroviral, adenoviral, herpes simplexviral, adeno-associated viral, polio viruses and vaccinia vectors.Examples of retroviral vectors include, but are not limited to, Moloneymurine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV)-derivedrecombinant vectors. More preferably, a non-human primate retroviralvector is employed, such as the gibbon ape leukemia virus (GaLV),thereby providing a broader host range than murine vectors, for example.Gene therapy vectors can be made tissue specific by, for example,linking the nucleotide sequence to a tissue-specific promoter. Multipleteachings of gene therapy are available for those skilled in the art,e.g., W. F. Anderson (1984) “Prospects for Human Gene Therapy” Science226: 401-409; S. H. Hughes (1988) “Introduction” Current Communicationsin Molecular Biology 71: 1-12; N. Muzyczka and S. McLaughlin (1988) “Useof Adeno-associated Virus as a Mammalian Transduction Vector”Communications in Molecular Biology 70: 39-44; T. Friedman (1989)“Progress Toward Human Gene Therapy” Science 244: 1275-1281 and W. F.Anderson (1992) “Human Gene Therapy” Science 256: 608-613.

The nucleic acid molecule can be delivered “naked” by direct injectioninto the blood stream or to the desired tissue or organ of a subject.Alternatively, the vector can be combined with a lipid compound whichfacilitates the uptake of the molecule by cells. The lipid compoundinclude liposome, lipofectins, cytofectins, lipid-based positive ions,and then introduced into the body fluids, the blood stream, or aselected tissue site. Liposome mediated gene therapy is well known inthe art and is described by, e.g., Lesoon-Wood et al., Human Gene Ther.6: 395, 1995; Tsan et al., Am. J. Physiol 268: 11052, 1995; Vieweg etal., Cancer Res. 5585: 2366, 1995; Trivedi et al., J. Neurochem. 64:2230, 1995; Hickman et al., Human Gene Ther. 5: 1477, 1994; Westbrook etal. Human Mol Genet. 3: 2005, 1994; Schmid et al., Z. Gastroenterol 32:665, 1994; Hofland et al., Biochem. Biophys. Res. Commun. 207: 492,1995; Plautz et al., Ann. N.Y. Acad. Sci. 7168: 144, 1994. Other DNAcarriers which can facilitate the uptake of a desired vector by thetarget cells include nuclear protein, or ligands for certain cellreceptors, which can be combined with a vector in engineered vesiclesfor delivery.

In another embodiment, the present invention provides a method oftreating a tumor in a subject by administering to the subject, atherapeutically effective amount of an antibody directed against Mts-1.

The present invention provides, as an example, that the administrationof an anti-Mts-1 monoclonal antibody to mice bearing highly metastaticCSML-100 tumors has a significant inhibitory effect on both tumor growthand metastasis.

Both monoclonal and polyclonal antibodies directed against a mammalMts-1 protein, including rat, mouse and human, can be employed forpracticing the methods of the present invention. Such antibodies can bereadily generated using the entire Mts-1 protein as an antigen or byusing short peptides, encoding portions of the Mts-1 protein, asantigens. Preferably, specific peptides encoding unique portions of theMts gene are synthesized for use as antigens for obtaining anti-Mts1antibodies. Those skilled in the art can refer to U.S. Pat. No.5,801,142 for relevant teachings. The Fab, Fab′, F(ab′)₂ fragments ofsuch antibodies, as well as single-chain anti-Mts-1 antibodies can alsobe employed in the present methods. The antibodies can be eitherunconjugated anti-mts-1 antibodies or anti-mts-1 antibodies conjugatedto a toxin.

In still another embodiment, the present invention provides methods oftreating a tumor in a subject by administering a therapeuticallyeffective amount of an antisense DNA of Mts-1 gene.

According to the present invention, antisense mts-1 nucleic acids caninhibit metastatic cancer by binding to sense mts-1 mRNA. Such bindingcan either prevent translation of mts-1 protein or destroy mts-1 sensemRNA, e.g., through the action of RNaseH.

An Mts-1 antisense DNA can have at least about 10 nucleotides,preferably, at least about 15 or 17 nucleotides, more preferably, atleast about 50 nucleotides. The antisense DNA is preferably insertedinto an expression vector in an operable linkage to a promoter which caneffect the transcription of the antisense RNA. Any of the foregoing genetherapy vectors can be used for practicing the methods of the presentinvention.

In practicing the above-described methods of the present invention, theactive compound (i.e., the binding-intercepting peptides, the nucleicacid molecules encoding such peptides, anti-Mts1 antibodies, or Mts-1antisense DNAs) can be used in combination with one another, or withother anti-tumor agents that are available in the art.

Another embodiment of the present invention relates to the animal tumorsand tumor cell lines developed in accordance with the present inventionwhich are useful as model systems of the metastatic process. Thesetumors and cell lines can be utilized for screening anti-metastaticdrugs and for developing therapeutic regimens for the treatment ofmalignant cancer is provided by the present invention. The tumorsprovided by the present invention include the IR6 and IR4 tumors. Thetumor cell lines provided by the present invention include CSML-0,CSML-50, CSML-100, HMC-0, HMC-Lr, T9, T36, LMEC, PCC4c-P, PCC4c-B,PCC4c-107, IR6CL₁, IR4 CL, ELCL₁, TRCL₁ and the murine lung carcinomaLine 1.

The tumors or cell lines of the present invention each have a highlypredictable metastatic potential; however the metastatic potentials ofrelated, but separate, tumors or cell lines can be very different. Theproperties, and metastatic potentials, of the tumors and cell lines ofthe present invention are fully described in Examples 1, 2, 3 and 12 andin Tables 1 and 2. While these tumors and cell lines were derived frommouse mammary carcinomas as well as rat thyroid and epithelialcarcinomas, they are useful for the development of a variety of humancancer therapies, for several reasons. First, cancer cells all havesimilar properties, including, for example, unrestrained growth and lackof contact inhibition, which suggests that the process of cancerdevelopment is similar in all cancers. Second, the morphologies andbiochemical properties of the tumors developed after injection of thesetumor-derived cells are identical to analogous tumors in humans. Hence,potential anti-cancer therapies or drugs may effectively be screened byemploying the animal model system of the current invention.

The utility of these unique tumors and cell lines is apparent to oneskilled in the art. Briefly, animals are injected with tumors ortumor-derived cells which have a predictable metastatic potential. Aproportion of the animals are treated with a potential anti-cancer drugor therapy. After a suitable period of time, all animals are sacrificedand the tissues of both treated and non-treated animals are examined forthe development of primary and secondary (metastatic) tumors. If atherapeutic regimen is successful, the 1 treated animals should have amuch lower incidence of tumor formation.

Both mouse and rat model systems are provided by the present inventionfor the development of cancer therapy. A spontaneous mouse mammarycarcinoma has been used to generate different cell lines with low,intermediate and high incidences of metastasis. This is done byintramuscular transplantation or subcutaneous tail transplantations ofthe original spontaneous mammary tumor cells into syngeneic mice.Intramuscular transplantation has yielded a cell line called CSML-0which has low metastatic potential. Solitary lung metastasis aredetected in less than 10% of CSML-0 injected animals sacrificed becauseof a moribund condition. The highly metastatic CSML-100 cell line hasbeen generated by selection of the metastatic phenotype throughsuccessive subcutaneous transplantations of CSML metastatic cells intothe tail. The CSML-50 cell line, selected during the generation ofCSML-100, has an intermediate level of metastatic potential.

A variety of rat tumors have been generated by irradiating normalFischer 344 rat thyroid cell suspensions and then transplanting thesecells into rats. Grafts of non-irradiated thyroid cells develop intomorphologically and functionally normal thyroid tissue aftertransplantation into Fischer 344 syngenic rats, if elevated levels ofthyroid stimulating hormone are also provided. Irradiation of thyroidcell suspensions before transplantation has produced a series of ratthyroid carcinomas which are histopathologically identical to humancounterparts. For example, the IR6 tumor, generated in accordance withthe present invention, is highly metastatic, while the IR4 tumor has lowmetastatic potential. Both tumors are structurally and histologicallyidentical to corresponding human tumors (FIG. 7).

The extensive variety of tumors and cell lines, and the varyingmetastatic potential of these tumors and cell lines, provides mouse andrat model systems amenable to carefully controlled studies directedtowards the dissection of the metastatic process. Therapeutic regimensfor treatment of malignant cancer can be developed by controlled studiesof groups of animals injected with cells of high, low and intermediatemetastatic potential. A drug, or pharmaceutical composition suspected ofhaving anti-metastatic potential, may be used to treat a proportion ofanimals from each group. The incidence of metastasis amongst the animalsreceiving the drug or pharmaceutical composition may be compared withthe incidence amongst animals not receiving treatment. Therefore, thepresent invention provides an animal system for distinguishing effectiveanti-metastatic drugs and therapies from those that are ineffective.

The Examples serve to further illustrate the invention without in anyway limiting same.

EXAMPLE 1 Materials and Methods

1. Medium

Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calfserum (FCS) was used for all cell lines. Cells were passed weekly.

2. Metastatic Activity

Metastatic activity was determined by intramuscular injection of 1×10⁴to 1×10⁶ tumor cells per tumor cell line in 10-15 mice. Either A/Sn orA/J mice were used.

For A/Sn mice, cultured tumor cells were trypsinized, rinsed andsuspended in sterile Hanks' salt solution. A total of 1×10⁶ cells in 0.3ml of Hanks' solution was injected subcutaneously into each 8 to 10 weekold A/Sn mouse. The mice were killed 4-5 weeks after tumor inoculationand the number of lung metastasis was counted. Non-metastatic cell lineswere defined as cell lines that did not result in visible metastases.Highly metastatic lines under the same conditions gave rise to multiplemetastases in target organs of each mouse.

Female A/J mice (4-6 weeks old) were injected either with 1×10⁴ cellsintravenously through the tail vein or with 1×10⁶ cells subcutaneouslyinto the abdomen. Fifteen days following intravenous injection and 4-6weeks after subcutaneous injection, the animals were sacrificed and thelung metastases were counted.

3. Mouse Tumor Cell Lines

CSML-0, CSML-50 and CSML-100 tumor cell lines were established inaccordance with the present invention from spontaneous mammaryadenocarcinomas of A/Sn mice. These cell lines are described in moredetail in Example 2.

HMC-0 and HMV-Lr are tumor cell lines which were also established fromspontaneous mammary adenocarcinomas of A/Sn mice. T-9, as well as T-36and its variant LMEC, are coupled sublines of two original tumors whichwere induced by ectopic transplantation of 6-7 day-old gestationsyngeneic embryos to CBA/J and A/Sn mice.

Cell lines, PCC4c-P, PCC4,-B and PCC4c-107 were derived fromPCC4-Blangy, PCC4-Pasteur and PCC4-107 teratocarcinomas, respectively.

A murine lung carcinoma, Line 1, cell line is highly metastatic, howeverwhen Line 1 cells are grown in the presence of the 3% DMSO, these cellslose their metastatic potential.

Some of the properties of the above cell lines, and their metastaticpotential, are described in Table 1.

TABLE 1 Metastatic Potential of Analyzed Mouse Tumors and Mouse TumorCell Lines Tumors and Spontaneous Target Cell Lines^(a) MetastasesOrgans Mammary carcinosarcoma CSML-0 low metastatic^(b) lung CSML-50 50%lung CSML-100 high metastatic^(c) lung Mammary Solid Carcinoma HMC-0 lowmetastatic liver^(d) HMC-Lr high metastatic liver^(d) Teratocarcinomacell line PCC4_(c)-B nonmetastatic — PCC4_(c)-P nonmetastatic —PCC4_(c)-107 nonmetastatic — C12- nonmetastatic — Embryocarcinoma, T-36node 50% lymph Cell line derived from T-36, T-36_(c) node 50% lymphEmbryocarcinoma, LMEV node high metastatic lymph Teratocarcinoma, T-9node low metastatic lymph Colon Adenocarcinoma, Acatol nonmetastatic —Melanoma, B-16 low metastatic lung Lung carcinoma; RL-67 high metastaticlung^(d) Lewis lung carcinoma, LLC high metastatic lung Murine lungcarcinoma cell Line 1: Grown without DMSO high metastatic Grown with 3%DMSO nonmetastatic ^(a)PCC4_(c)-B, PCC4_(c)-P, and PCC4_(c)-107 are celllines derived from PCC4-Blangy, PCC4-Pasteur, and PCC4-107teratocarcinomas. ^(b)Low metastatic indicates 20% of injected mice giverise to solitary metastases. ^(c)High metastatic indicates 100% ofmultiple metastases in target organs. ^(d)Metastases in other organs.

3. Rat Tumors and Rat Tumor Cell Lines

An established epithelial cell line, FRTL5, was derived from a cultureof rat thyroid cells and is not tumorigenic. In accordance with thepresent invention, two tumorigenic but non-metastatic derivatives ofFRTL5 cells, ELCL.sub.1 and TRCL.sub.1, have also been isolated. Theproperties of these non-metastatic cell lines are further elaboratedupon in Table 2 and in Example 3.

The IR6 tumor is a radiation induced, transplantable anaplastic thyroidcarcinoma, of epithelial origin. It is a poorly differentiated, highlyaggressive adenocarcinoma which is highly metastatic. IR4 is anothertransplantable, radiation induced thyroid tumor which is moderatelydifferentiated and has low metastatic potential. The properties of thesetumors are further elaborated on in Example 3 and in Table 2.

4. Nucleic Acid Purification and Analysis

Tumor cells were cultivated and prepared for subcutaneous injection intomice as described under the metastatic activity subsection of thissection. Injected mice were examined weekly for the appearance oftumors. Tumors were excised and used for DNA and RNA preparations. TotalDNA was prepared from cells according to Sambrook et al. (MolecularCloning: A Laboratory Manual. Cold Spring Harbor, Vol. 2, LaboratoryPress, 1989. Pages 9.1-9.62).

RNAs were prepared from different tumor cells and normal cells accordingto the procedure described by Chomczynski et al. (1987, Anal. Biochem.162: 156-159) or Sambrook et al. (Molecular Cloning: A LaboratoryManual, Vol. 1, Cold Spring Harbor Press, 1989:7.1-7.87). Gelelectrophoresis of RNA, RNA blotting to nylon membrane filters, andhybridization with nick-translated DNA probes was as described inGrigorian et al. (1985, EMBO J. 4: 2209-2215).

Southern blots were performed using 10 .mu.g of genomic DNA extractedfrom mouse liver, CSML-100 cells, human placenta and liver, rat liver,pig liver, and chicken liver. DNAs were digested with BamHI, EcoRI, andPstI endonucleases. Following electrophoresis in a 0.8% agarose gel, theDNA was transferred onto a nylon membrane (Hybond N. Amersham). Thefilter was prehybridized and hybridized following the standard procedureof Sambrook et al., supra.

EXAMPLE 2 Development of Benign and Metastatic Mouse Tumor Cell Lines

CSML-0, CSML-50 and CSML-100 are tumor lines established fromspontaneous mammary adenocarcinomas of A/Sn mice. CSML-0 was derivedfrom a tumor maintained by intramuscular passages and was characterizedas having a low metastatic potential. Solitary lung metastases weredetected in less than 10% of autopsied animals that had been killedbecause of a moribund condition. A second, highly metastatic subline,CSML-100, was developed by selecting for a metastatic phenotype insuccessive transplantations (via successive subcutaneous tailinjections) of initially rare, and subsequently more frequent, CSMLmetastatic tumor cells. The frequency of metastasis to the lung byCSML-100 cells was 100%, by any route of primary inoculation. CSML-50represents a cell line with an intermediate level of metastaticpotential which was developed during the establishment of CSML-100. Thefrequency of lung metastasis by CSML-50 cells was about 50%.

The CSML-100 tumor line also caused tumors to form in A/J mice (Jacksonlaboratories) which have a similar genotype to that of A/Sn mice. CSMLcells were not rejected by A/J mice and metastases were detected inlungs and other organs by any injection route.

A/J mice intravenously injected with CSML-100 developed tumors within6-7 days of injection. Even when only 1×10⁴ CSML-100 cells wereinjected, abundant metastases were found in lungs by 15 dayspost-injection (FIG. 9c). A/J mice injected with CSML-100 cells by thesubcutaneous route had approximately 250 spontaneous metastases per lung4-6 weeks later (FIG: 9 d). Mice injected with CSML-0 by either route ofinjection had only 10-25 tumors per lung (FIGS. 9a and 9 b). Aftersacrifice of each mouse, the ovaries, liver, kidney, gonads, muscle, andbrain tissues were preserved for immunohistochemical analysis. Suchanalysis indicated mts-1 was highly expressed in metastasized tumors,particularly in the ovarian and lung tumors.

EXAMPLE 3 Development of Benign and Metastatic Rat Tumors and Rat TumorCell Lines

A number of rat thyroid carcinomas and cell lines have been developed inconjunction with the present invention, by irradiating normal Fischer344 rat thyroid cell suspensions before transplantation into the rat.Grafts of non-irradiated, monodispersed rat thyroid cells develop intomorphologically and functionally normal thyroid tissue within a shorttime after transplantation into Fischer 344 syngeneic rats, if the levelof thyroid stimulating hormone (TSH) within the rat is elevated byinjection of TSH. If thyroid cells are irradiated beforetransplantation, thyroid carcinomas develop. The IR6 tumor was obtainedas a radiation induced, transplantable anaplastic thyroid carcinoma ofepithelial origin. IR6 was found to be poorly differentiated, highlymetastatic and did not require TSH for growth. The IR4 tumor was alsoobtained as a radiation induced rat thyroid carcinoma but IR4 ismoderately differentiated into a follicular carcinoma, grows slowly onlywhen TSH is provided and has low metastatic potential. IR6CL.sub.1 is acell line derived from the IR6 tumor which retains the originalproperties of the parent IR6 tumor, e.g., it grows independently of TSH,is poorly differentiated and is highly metastatic.

An established epithelial cell line, FRTL5, derived from a culture ofrat thyroid cells was also obtained. FRTL5 cells requires TSH andremains highly differentiated, but produces no tumors when injectedsubcutaneously into syngeneic Fischer 344 rats. Two tumorigenicderivatives of the FRTL5 cell line, ELCL₁ and TRCL₁, have also beenisolated and characterized. ELCL₁ was obtained as a spontaneous mutantof FRTL5, and subsequently established as a transformed cell line whichrequired low levels of TSH for growth. ELCL₁ formed primary tumors uponsubcutaneous injection in syngenic rats but no metastasis was observed.TRCL₁ was a radiation induced mutant of FRTL₅ which was then establishedas a transformed cell line with no TSH requirement for growth. TRCL₁cells produced fast-growing primary tumors with little or no potentialfor metastasis.

Some of the properties of the above described tumors and cell lines aresummarized in Table 2.

TABLE 2 Metastatic Potential of Rat Tumors and Rat Tumor Cell LinesTumors and Spontaneous Target Cell Lines Metastases Organs Thyroidcarcinoma Lung, 1R6 tumor high metastatic Liver, 1R4 tumor lowmetastatic Kidney Thyroid cell line FRTL5 (non-tumorigenic)nonmetastatic ELCL₁ (tumorigenic) nonmetastatic TRCL₁ (tumorigenic)nonmetastatic

EXAMPLE 4 Isolation of the Murine mts-1 Gene

mRNA from CSML-100 and CSML-0 cell lines was prepared as described byChomczynski et al. supra, and polyadenylated mRNA was selected as inSambrook et al. (Molecular Cloning: A Laboratory Manual, Vol. 1, ColdSpring Harbor Laboratory Press, 1989. Pages 7.1-7.29). 2 μg poly (A)⁺mRNA from highly metastatic CMSL-100 cells was treated with reversetranscriptase under conditions appropriate to generate a single strandedcomplementary DNA (cDNA) (Sambrook et al., supra. Vol. 2. Pages8.1-8.86). This CMSL-100 cDNA pool was subjected to subtractivehybridization with 50 μg poly (A) mRNA from low metastatic potentialCMSL-0 cells to remove cDNA's with no role in the development ofmetastasis. The cDNA/RNA mixture was heated at 100° C. for 5 min.,cooled on ice and placed in a final reaction volume of 1 ml in 7% phenol(adjusted to pH 7.6 with 0.1M Tris-HCl, 1.25M NaCl, 120 mM sodiumphosphate buffer, pH6.8) in a 10 ml glass centrifuge tube in. The tubewas shaken for 7 days at 25° C. After hybridization, the mixture wasextracted twice with chloroform, dialyzed against 10 mM Tris-HCl (pH7.5), 1 mM EDTA to remove excess salts, and then precipitated withethanol. Double stranded cDNA/mRNA, representing functions which are notunique to the metastatic phenotype, were removed by passage through ahydroxyapatite column. The single stranded cDNA was made double strandedand cloned into a λgt10 vector by standard procedures (Sambrook et al.,supra pages 8.1-8.86). Functions expressed highly during metastasis weredetected by differential hybridization with CSML-100 and CSML-0P-labeled cDNA probes. Mouse mts-1 cDNA clones were identified asstrongly hybridizing with the DCSM-100 probe but weakly hybridizing withthe CSML-0 probe.

EXAMPLE 5 Isolation of a Rat mts-1 cDNA

Rat cDNA libraries were prepared from normal thyroid and radiationinduced thyroid carcinoma tissues as well as cell lines derived fromnormal and carcinogenic thyroid tumor cells. Poly(A)⁺ mRNA was purifiedfrom highly metastatic IR6 tumors and from low metastatic potential IR4tumors. Single-stranded cDNA was synthesized from IR6 poly(A)⁺ RNA andthe IR6 mRNA was hydrolyzed. This IR6 cDNA pool was subjected tosubtractive hybridization with a 50-fold excess of IR-4 poly(A)⁺ mRNAaccording to the phenol emulsion reassociation technique (PERT method)of Kohne et al. (1977, Biochemistry 16: 5329-5341). Single strandedcDNA, representing functions likely to be involved in the metastaticphenotype, was isolated from the subtractive hybridization mixture bypassage through a hydroxyapatite column (which will bind double strandednucleic acids, i.e. the RNA:DNA hybrids representing the IR6 functionsof low metastatic potential) followed by alkaline hydrolysis of theremaining IR4 mRNA. The single-stranded cDNA pool was made doublestranded and cloned into a λgt10 cloning vector.

The subtracted IR6 cDNA library was screened differentially with³²P-labeled single stranded cDNA probes generated by treatment of IR6and IR4 poly(A)⁺ mRNA with reverse transcriptase. mts-1 clones wereidentified by strong hybridization with the IR6 probe but weakhybridization with the IR-4 probe.

EXAMPLE 6 Isolation of the Human mts-1 cDNA

A human cDNA library was constructed in λgt10 using poly (A)⁺ RNAprepared from HeLa cells. The library was screened with a ³²P-labelledmouse mts-1 cDNA probe at 420 in 50% formamide. Filters were washed in2×SSC with 0.1% SDS at room temperature and then twice in 0.2×SSC with0.1% SDS at 50° C. Strongly hybridizing cDNA clones were sequenced; thehuman mts-1 cDNA was identified by high sequence similarity to the mousemts-1 cDNA in regions outside the highly conserved Ca⁺⁺ binding domain.This human mts-1 clone is full length as judged by sequencing of thehuman genomic mts-1 gene and by primer extension analysis of mts-1 mRNAusing mts-1 oligonucleotide probes. The nucleotide and amino acidsequences of the human mts-1 gene are provided as SEQ ID NO: 1 and 2,and also given in FIGS. 1 and 2.

The mts-1 cDNA was also isolated from human melanoma cell line Wm64 byreverse transcription of mRNA isolated from those cells followed bypolymerase chain reaction.

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

Total RNA from human melanoma cell line Wm64 was pretreated with RNasefree DNase I (1 U/μl) in 2 mM MgCl₂ for 30 minutes at 37° C. then 95° C.for 5 minutes to inactivate the DNase; poly A⁺ RNA was not routinelytreated with DNase I before an RT-PCR experiment. RNA (1 μg total RNA or50 ng poly A ⁺RNA) was reverse transcribed in the presence of 50 mMTris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl₂, 3 μM oligo-dT₁₅, 0.3 mM eachdNTP, 200 U M-MLV reverse transcriptase at 22° C. for 10 minutes, 42° C.for one hour and 90° C. for 10 minutes. The following human mts-1primers were used for synthesis of the human mts-1 cDNA by PCR reaction:

5′ ATG GCG TGC CCT CTG GAG AAG-3′ (SEQ ID NO:8)

5′ TTT CTT CCT GGG CTG CTT ATG-3′ (SEQ ID NO:9).

PCR amplification was done in 10 mM Tris HCl (pH 8.4), 50 mM KCl, 1.5 mMMgCl₂, 0.01% gelatin with 2.5 mM dNTP, 0.6 μm each primer and 1.25 U TaqDNA polymerase. Amplification was by 35 cycles of: 94° C. for 1 min; 52°C. for 2 min; 72° C. for 3 min, followed by a 7 min extension period at72° C.

Amplified DNA was isolated from a 1% agarose gel an cloned into abaculovirus transfer vector as described in Example 7.

EXAMPLE 7 Expression of the mts-1 Gene Product

Overexpression of the mts gene product, is accomplished by DNAtransfections using the vector system described by Lockshon andWeintraub. This vector is a pUC19 based vector system, very similar tothe Bluescript.™. vector (FIG. 3). In the unique HindII site of theBluescript.™. vector, a eukaryotic control element harboring a strongmurine sarcoma virus promoter, followed by a unique EcoRI site, followedby SV40 polyadenylation sequences is introduced. The complete mts-1 cDNAis introduced into the unique EcoRI site downstream from the MSV-LTRsequences. Because of the presence of an internal EcoRI site in themts-1 cDNA, partial EcoRI digestion of the mts-1 recombinant is done toisolate the entire mts-1 cDNA molecule. Retroviral promoters with LTRsare very strong and overexpression of the mts transcript is expected.The mts-1 recombinant expression vector can be used for both permanentor transient expression. However, stable (permanent) transfectants aredesirable because stable transfectants can be clonally purified, andrepresent a homogeneous population of a given phenotype useful forquantitating metastatic potential.

Expression of mts-1 Protein from a pTrcHisB Vector

Large quantities of the mts-1 specific protein were expressed using theinducible bacterial vector pTrcHisB (Invitrogen) (FIG. 10a).Murine.mts-1 cDNA was subcloned in frame (confirmed by sequenceanalysis) into a BamHI-KpnI site with the multiple cloning site ofpTrcHisB. This generated plasmid pTBM1. The fusion protein expressed bypTBM1 had 6 tandem histidine residues (which have a high affinity forNi⁺⁺ charged resin), an enterokinase specific cleavage site, and themts-1 protein product. Expression of the fusion protein encoded by pTBM1was induced by IPTG. Similar constructs were generated with human mts-1cDNA.

Expression of mts-1 Protein in a baculovirus Expression Vector

A plasmid containing the cytomegalovirus promotor was used to constructpCMV/mts-1_(h) or pCMV/mts-1_(m) high expression vectors harboring mts-1human and murine cDNAs, respectively.

The baculovirus expression vector mts-1-BacPAK₆ plasmid was constructedfrom the pCMV clones as follows. pCMV-mts-1 was digested with BamH1, andthe mts-1 cDNA fragment was purified from a 1% agarose gel. The purifiedfragment was ligated into BamH1-cleaved pBacPAK1 and the ligation mixwas transformed into E. coli JM109 cells. Positive clones wereidentified and plasmid DNA was sequenced to confirm the orientation andintegrity of the ligation junction.

Transfer vector pBacPAK-mts-1 was transfected into Sf21 cells, alongwith Bsu361 digested BacPAK6 viral DNA. Soon after infection, the cellswere overlayered with 1% agarose to visualize the plaques and to preventmixing of clones. After 4-5 days of infection, the cells were stainedwith neutral red which is taken up by healthy cells, but not by the deadcells. Plaques appeared as clear circles against red or pink background.Western Blot analysis using the α-mts-1 antibody was conducted toconfirm that several mts-1 recombinant viruses produced mts-1 proteins.

EXAMPLE 8 Purification of mts-1 Protein

Purification of the mts-1 protein parallels that of other S100 familymembers which have been purified to homogeneity from bovine brain(Baudler, et al. J. Biol. Chem. 261: 8204-8212, 1986). Exceedingly highdegrees of purification can be achieved because of the stability of theprotein and the availability of several affinity chromatography stepsincluding phenothiazine-agarose, zinc dependent binding to phenylsepharose. FPLC chromatography on Mono Q is known to separate S100family members and other HPLC columns have been developed such asmelittin silica, to affinity purify S100 proteins. Tissues or cellsproviding large amounts of mts-1 include not only the bacterial, yeastand mammalian cell lines engineered to express large quantities ofrecombinant mts-1, but also the highly metastatic tumors and cell linesshown to express mts-1 by the present invention.

Purification of His-mts-1 Fusion

An overnight culture transformed with pTBM1 was diluted 1:100 andallowed to grow 1.5 hours (until OD₆₀₀=0.3). The culture was theninduced with 1 mM 1PTG and allowed to grow 4.5 hours more at 37° C.Cells were harvested, cell pellets were then collected by centrifugationand resuspended in a 6M guanidinium-HCl buffer. The cells were stirredfor 1 hour and then centrifuged at 18K rpm for 15 min at 4° C. Thesupernatant was collected and added to a 50% slurry of Ni⁺⁺ NTA resin(obtained from Qiagen). The mixture was stirred for an hour and loadedonto a column. The column was washed in a series of urea based bufferswhich differed only in pH (each wash being of a lower pH). The proteinwas eluted in 3 ml fractions using buffer D (8M urea, 0.1M Na Phosphate,0.01M Tris/HCl, pH 5.9). A large amount of the protein did not eluteuntil the pH of the buffer was lowered to 4.5 (buffer E): monomericforms of the histidine fusion eluted in buffer D, whereas aggregateseluted in buffer E. Aliquots of each fraction were boiled in SDS-PAGEloading buffer and loaded onto 12% SDS-polyacrylamide gels. The gelswere stained with Coomassie Brilliant Blue. The results of suchexperiments are depicted in FIG. 10b. After the purity of the His-Mts1fusion protein was confirmed, assays were performed to determinerelative protein concentrations. Fractions D2 and E1 (which containedapproximately 3.2 mgs protein total) were pooled and run on anotherSDS-polyacrylamide gel. Strips were cut out from the gel and stained inCoomassie Brilliant Blue to determine the location of the His-Mts1protein in the gel. The portion of the gel containing the His-Mts1fusion was cut out and the protein was isolated from the gel by elution.

EXAMPLE 9 Generation of Polyclonal Antibodies

Antibodies Against mts-1 Peptides

Synthetic oligopeptides with the following amino acid sequences weremade:

1) Human mts-1 amino acids 2-11 (unique):Ala-Cys-Pro-Leu-Glu-Lys-Ala-Leu-Asp-Val

2) Human mts-1 amino acids 22-37 (the calcium binding domain):Lys-Glu-Gly-Asp-Lys-Phe-Lys-Leu-Asn-Lys-Ser-Glu-Leu-Lys Glu-Leu

3) Human mts-1 amino acids 42-54 (unique):Leu-Pro-Ser-Phe-Leu-Gly-Lys-Arg-Thr-Asp-Glu-Ala-Ala

4) Human mts-1 amino acids 87-101 (unique):Asn-Glu-Phe-Phe-Glu-Gly-Phe-Pro-Asp-Lys-Gln-Pro-Arg-Lys-Lys

Peptides 1, 3 and 4 were chosen as mts-1 antigens because they encodeunique proteins of the mts-1 protein, i.e. these regions of the mts-1protein do not share homology with other proteins, in particular withother calcium binding proteins. Peptide 2 was chosen because it encodesthe calcium binding domain of mts-1. Therefore, peptide 2 generatesantibodies reactive with many members of the calcium binding proteinfamily.

New Zealand white female rabbits were immunized by subdermal injectionwith 100 μl of Freund's complete adjuvant containing 0.1-1 mg ofoligopeptide in 10 locations along the back. The rabbits were firstshaved on both sides of the back for easy subdermal injection. Theantigen-adjuvant mixture was prepared by mixing in two connected 1 mlglass tephlon syringes. Typically rabbits are then injected with abut 1mg of antigen at each 2 month interval following the primary injection,until the serum is positive at a dilution of greater than 10⁻⁴ whenassayed by immunoblotting.

Antibodies Against Whole mts-1 Protein

The mts-1 protein was expressed as a His-Mts1 protein (Examples 7 and8). Host cell lysates containing the His-Mts1 protein were fractionatedover a Ni⁺⁺ NTA column (Qiagen). Fractions containing the most His-Mts1protein were pooled and electrophoresed on an SDS-polyacrylamide gel.The purified protein was eluted from the gel and sequenced to confirmthat it was His-Mts1.

Three chickens were then immunized with the purified His-Mts1 protein.Chickens were chosen for two reasons. First, mts-1 is highly conservedin mammals and an avian system was expected to provide a better immuneresponse. Second, antibodies can easily be obtained from the eggs of thechickens. Continuous bleeding of the animal to obtain antibodies is,therefore, avoided.

The polyclonal antibody generated was named α-mts-1, and its efficacy onWestern blots and tissues was established (see Example 16 and FIGS. 13and 14).

EXAMPLE 10 Monoclonal Antibody Production

Monoclonal antibodies are prepared in accordance with the techniquesdeveloped by Kohler and Mulskin (Eur. J. Immunol. 6:511-519, 1976) andHarlow et al. (Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, 1988). Balb/c mice are immunized subdermally with 100μl of Freund's complete adjuvant containing 0.1-1 mg of the conjugatedor non-conjugated mts-1 oligopeptdies described in Example 9. Two weeksafter the initial injection, the mice are boosted with the appropriatemts-1 antigen by intravenous and intraperitoneal injection of 100 ug ofantigen in phosphate buffered saline (PBS).

Five days after the last injection and after confirmation of thepresence of antibody in mouse sera, the mice are sacrificed and theirspleens removed. Spleen cells are obtained by gentle disruption of thespleen in a 7 ml Dounce homogenizer in 3.5-4 ml PBS. The cells are thenpelleted at 1200 rpm in a PR6 centrifuge for 6 minutes at roomtemperature. The supernatant is removed into a suction flask, and thecells are resuspended in 15 ml 0.83% NH₄ Cl. This suspension isincubated at room temperature for 5 minutes then underlain with 10 mlfetal calf serum at 37° C. The cells are again pelleted bycentrifugation for 8 minutes, at 1200 rpm at room temperature, then thesupernatant is withdrawn into a suction flask cells resuspended in 20 mlPBS.

The following solutions are prepared for use in the subsequent cellfusion:

Hypoxanthine (H), 680 mg/100 ml H₂O; add 204 drops conc. H₂SO₄y; heat todissolve Aminopterin (A), 46.4 mg/100 ml H₂O; add 2 drops 1.0N NaOH todissolve Thymidine (T), 775 mg/100 ml H₂O; add 45 mg glycine;

PEG-DME—melt PEG at 42° C., then add 1 ml DME (at 37° C.); adjust pHwith 1.0N NaOH to 7.6;

DMEM—-to 500 ml DME add 37.5 ml a−horse serum; 37.5 ml FCS, 10.0 mlL-glutamine, 0.2 ml garamycin;

2×HAT-DME—to 200 ml DME add 25.0 ml a −horse serum, 25.0 ml FCS, 4.0 mlL-glutamine, 0.2 ml garamycin, 0.8 ml H, and 0.8 ml A, and 0.8 ml T(2×HT-DME omits A);

Cloning Agar—350 mg unwashed Difco agar in 25 ml H₂O, autoclaved;

Cloning Medium—to 25 ml 2×DME, add 35 ml filtered, condition DMEM, 7 mla −horse serum, 7 ml FCS, 1 ml L-glutamine, 0.1 ml garamycin.

Two 30 ml flasks of plasmacytoma P3 NS1/1-Ag4-1 cells are added tocentrifuge tubes and spun down at 1200 rpm for 8 minutes at roomtemperature. The spleen cells are resuspended in 20 ml PBS. From eachsuspension, 0.01 ml is removed and added to 0.1 ml 0.4% trypan blue and0.3 ml PBS and the cells counted. The volume of each suspension isadjusted so as to obtain a spleen cell to NS1/1-Ag4-1 cell ratio of10:1, and the suspensions are then mixed. The mixture is pelleted at1200 rpm for 8 minutes at room temperature and all but about 0.1 ml ofsupernatant removed. The cells are then resuspended in the remainingliquid and then added to 1.3 ml of 1:1 PEG-DME solution, pH 7.6. Everyminute the volume of the solution is doubled with DME until the finalvolume is 25 ml.

The cells are again pelleted, the supernatant decanted, and the cellsresuspended in enough 50% 2×HAT-DME/50% conditioned DMEM (thesupernatant retained form the Sp2/0 cells above) to yield a finalconcentration of about 3.5×10⁶ spleen cells. The cells are distributedinto a 96-well flat-bottom microtiter plate (TC-96; Flow Laboratories),at 0.1 ml/well. The plate is incubated at 37° C. in humidified air/CO₂until visible colonies appear, usually about 10-12 days. The contents ofthe well is transferred to 0.5 ml of HAT-DME/conditioned DME in a TC-24plate (Flow Laboratories). When healthy cell growth appears (about 2-5days), about 0.35 ml medium is removed and tested for antibodyproduction by enzyme-linked immunosorbent assay (ELISA), hemagglutinininhibition assay, or neuraminidase inhibition assay. When those cellsproducing the antibodies of interest are growing well, one drop for eachculture is transferred into 1.0 ml DMEM in a TC-24.

To clone the hybrid cells, 25 ml of melted agar and 76 ml of cloningmedium is combined, and 5 ml is pipetted into 60 mm petri dished andleft to solidify. Cells from DMEM cultures are diluted in 50% DMEM/50%conditioned DMEM, 10⁻¹ or 10² depending on cell growth. Into steriletubes is placed 0.1 ml of each of the two dilutions, and to each isadded 0.9 ml of cloning medium/agar mixture. This is mixed well andpoured over the surface of the agar underlay. After solidification theplates are incubated at 37° C. incubator until colonies are visible withthe naked eye, typically about 7-10 days. Colonies are then picked andtransferred 0.1 ml of DMEM/conditioned DMEM in a TC-99 plate andincubated at 37° C. in a CO₂ incubator. After the culture is acidic(usually 1-4 days), transfer is made to 0.05 ml DMEM in TC-24 plate.When the growth is 50% confluent, the medium is removed and tested forantibody production are previously. Those clones producing mts-1specific antibodies are moved into 5 ml DMEM in 25 cm² flasks. Clonedcells are then frozen or injected into mice for ascites production.

EXAMPLE 11 Sandwich Assay for mts-1

For detection of the presence of mts-1 in serum or cleared cell lysatesof tissue specimens, approximately 100 ul of a monoclonal antibodyprepared as in Example 9 or 10 is immobilized on latex beads and iscontacted with about 100 ul of the serum or cleared lysate to be tested.The immobilized antibody and lysate are allowed to react for a period ofabout ten minutes and then the latex beads with the mts-1 antigen boundto the immobilized antibody are rinsed with a solution of PBS (phosphatebuffered saline). To the latex beads is then added about 100 ul of mts-1specific antibody conjugated to horseradish peroxidase. The labeledantibody bead mixture is incubated for a period of about ten minutes. Atthis time, an enzyme substrate, hydrogen peroxide and aminoantipyrine,are contacted with the beads, and this mixture is incubated for a periodof about 5-10 minutes, at which time the development of color in thesample is an indication of a positive reaction and the presence ofmts-1.

EXAMPLE 12 Expression of mts-1 is 10-100 Fold Higher in Metastatic TumorCells than in Non-Metastatic Cells

To examine the expression levels of mts-1, mRNA was purified frommetastatic and benign tumors, and cell lines derived from such tumors,as well as from corresponding normal tissues. Purified RNA was sizefractionated in a gel and blotted onto nylon membranes for Northernanalysis with mts-1 nucleic acid probes.

FIG. 4 shows that the CSML-0 cell line of the present invention, whichhas a very low metastatic potential, had very low, or non-detectablelevels of the mouse mts-1 transcript. In contrast, the CSML-100 cellline of the present invention, which has an extremely high metastaticpotential, expressed high levels of mts-1. It is estimated thatmetastatic CSML-100 cells express at least 100-fold more mts-1 than donon-metastatic CSML-0 cells.

Similarly, in a separate experiment, various metastatic andnon-metastatic tumors and tumor cell lines were tested for their mts-1expression levels, by Northern analysis using a ³²P-labeled mouse mts-1probe. The properties of these tumors and cell lines are described indetail in Examples 1, 2 and 3 and in Tables 1 and 2. As shown in FIG. 5,only those tumors and cell lines which are metastatic (indicated by an“M” above the gel lane) exhibit high levels of mts-1 expression.Metastatic cell types exhibiting increased mts-1 expression include:RL-67 lung carcinoma tumors, Lewis Lung carcinoma. tumors, LMECembryo-carcinoma tumors, and T-36 embryo-carcinoma tumors and celllines.

FIG. 6 shows that the highly metastatic adenocarcinoma rat tumor, IR6(lane 5), and a cell line derived from IR6 (lane 7), as well as ametastatic cell line derived from a mouse lung carcinoma, Line 1 (lane3) all exhibit 10-100 fold increased levels of mts-1 expression comparedto a tumorigenic but non-metastatic cell line, TRCL₁ (lane 6) or anon-tumorigenic FRTL5 cell line (lane 8).

Hence these data demonstrate unequivocally that mts-1 expression isincreased 10-100 fold in metastatic cells of diverse types relative tonormal cells or non-metastatic (benign) tumor cells.

Table 3 further illustrates that only metastatic cells or cells with ahigh degree of motility express high levels of mts-1 RNA. Detection wasby northern analysis using γ-actin expression for normalization.Autoradiograms were densitometrically traced, and a numerical valuebetween 0-5 was assigned relating the tracing peak height to the amountof expression. The status of each cell type tested was characterized asnormal (N), benign (B), metastatic (M) or cell line (C). The number ofsamples tested is indicated under the status of cell type.

Table 3 illustrates that only metastatic cell types have an mts-1expression level greater than 0.5. Accordingly, high levels of mts-1expression are observed in numerous metastatic cell types including, forexample, liver hepatomas, lung carcinomas, pancreatic cancers, breastadenocarcinomas, endometrial cancers, ovarian cancers, cervical cancers,melanomas, lymphomas and leukemias. However, such high levels of mts-1expression are observed only in metastatic cells, non-metastatic cellsdo not express high levels of mts-1.

TABLE 3 Selective Expression of mts-1 in Metastatic Cells or Cells withHigh Degree of Motility No. of Samples; Status Level Phenotype of TissueN B M C of Expn. Adult Liver 5 — — — 0 Liver Adenoma — 5 — — 0 LiverHepatoblastoma — 5 — — 0 Liver Hepatoma — — 4 — 1.0 Adult Colon 4 — — —0.4 Colon Carcinoma — 5 — — 0.43 Adult Kidney 2 — — — 0.1 KidneyCarcinoma — 2 — — 0.1 Adult Lung 2 — — — 0.1 Small Lung Carcinoma — — 2— 1.0 Adult Pancreas 1 — — — 0 Pancreatic Cancer — — 1 — 1.0 NormalBreast 4 0 Breast Carcinoma 2 Breast Adenosarcoma — — — 2 1.0Endometrial Cancer — — 2 — 1.5 Ovarian Cancer — — 2 — 1.4 CervicalCancer — — 2 — 1.5 ASPC 1 Pancreastic Cancer 1M 1.0 AN3CA Endometrial 1M1.5 Cancer BIX3A Ovarian Cancer 1M 1.5 Hela Cervical Cancer 1M 1.5MCF7-1 Breast Cancer 1 0 AS49-1 Lung Cancer 1(M) 0.8 MC1 NeuroblastomaLine 1 0 Y79 Retinoblastoma 0 Primary Melanoma Wm278 1 0.5 Corcl PrimaryMelanoma 0 1.0 Wm8 Melanoma 1(M) 2.0 Wm164 Melanoma 1(M) 2.0 Normal BCells Do Not Express mts-1 B-Cell Lymphoma & Leukemias Type No. Level ofExpn. Cleaved B Cell Lymphoma 1 3 Hairy Cell Leukemia 1 4 CML Crisis BCells 3 3 No. of Average Leukemias Samples mts-1 Expn. Type Tested LevelCML (chronic probe) 23 0.49 CML (crisis) 12 1.9 CMML 1 1.0 ALL 1 3.0 AML6 0.7 AMML 2 1.0 Pure Monocytic Leukemia 3 1.5 Abnormal BloodInfiltrated with High WBC Count Separated by Ficoll-Hypague GradientPellet 4 0.3 Interface 5 0.6

EXAMPLE 13 Introduction of the mts-1 Gene into Cultured Cells Confers aMetastatic Phenotype

According to the present invention, mts-1 is not expressed in normal, ornonmetastatic tumor cell lines, from the rat thyroid or the mouse lung.However, the highly metastatic Line 1 cell line, derived from a mouselung carcinoma, does express mts-1 mRNA. When Line 1 cells are grown inthe presence of 3% DMSO, they lose their metastatic potential and alsodo not show detectable levels of mts-1 mRNA. These data indicated thatmts-1 expression is correlated with the metastatic phenotype.

To establish that high levels of mts-1 expression can confer ametastatic phenotype the rat mts-1 cDNA was cloned into the MSV vectordepicted in FIG. 3, to allow high expression of the mts-1 protein. Thismts-1 expression vector was co-transfected into mouse lung carcinomaLine 1 cells with a plasmid encoding a selectable neomycin (Neo) gene.Stable cell lines resistant to neomycin were tested for integration ofthe mts-1 gene into their genome by Southern blot analysis of theirgenomic DNA. The controls for this experiment were Line 1 cells stablytransfected only with the selective neomycin resistance gene grown inthe presence of 3% DMSO, as well as non-transfected Line 1 cells grownwithout DMSO.

Ten transfectants (N1-N10) possessing the transfected mts-1 gene weregrown in 3% DMSO to test whether acquisition of the highly expressedrecombinant mts-1 gene could generate a metastatic phenotype in cellsthat are normally not metastatic. 10⁵ cells of transfectants N2, N3, N4,N5, and N8 were injected in the tail veins of 3 mice. As controls, 10⁵cells of Line 1 cells, and two neomycin only transfectant cell lines(Neo 2 and Neo 3) were injected into the tail veins of 3 mice. Theanimals were sacrificed after 2 weeks and tested for lung metastasisafter staining with India ink and fixation. The animals injected with N4and N5 cells grown in 3% DMSO prior to injection, exhibited high levelsof metastasis, equivalent to Line 1 cells grown in the absence of DMSO,while other cell lines gave rise to low levels of metastasis. The factthat not all transfected cell lines gave rise to high levels ofmetastasis might have been due to a variation in mts-1 expression levelscaused. by mts-1 insertion into “silent” regions of the genome. Toexamine the expression levels of mts-1 in N1-N10 transfectants grown in3% DMSO, mRNA was extracted from these cell lines prior to injectioninto mice, and analyzed for mts-1 mRNA expression levels by Northernanalysis. As shown in FIG. 8, not all transfectants exhibit high levelsof mts-1 expression, probably because of the influence of genomicregulatory elements lying near the mts-1 insertion site. Transfectantcell lines N3, N4 and N5 have high levels of mts-1 expression, but theN3 cell line gives rise to a low molecular weight mts-1 transcript,indicating that the mts-1 gene of this transfectant cell line may bedefective due to a rearrangement during transfection and integrationinto the genome.

Table 4 shows that similar data were obtained by intravenous injectioninto rats of transfectant cell lines containing expression vectors withthe rat mts-1 gene in a sense and antisense orientation, relative to theMSV LTR promoter.

Hence, these data indicate that the metastatic phenotype can begenerated in non-metastatic cells by the introduction of a highlyexpressed mts-1 gene.

TABLE 4 Mouse Lung Metastasis Counts Using Different mts-1 TransfectantsClone Clone Clone 156/3 156/4 156/5 Neo+ Line I+ (N₃)+ (N₄)+ (N₅)+ DMSODMSO Line I DMSO DMSO DMSO Intravenous 5 57 190 342 355 360 Injection 038 205 300 495 460 10⁵ Cells 0 65 251 320 310 310 Into Tail 11 68 300 75 142 120 Vein rat mts1 clone 156 = sense construct rat mts1 clone 162= antisense construct

In the above experiment IR6 tumor cells alone generate lung metastasisin 20% of the injected rats, with 1-2 tumors observed in the kidneys ofsome rats. 50% of rats injected with transfectants containing mts-1 in asense orientation (cell lines 156/2, 156/7 and 156/8) had metastases,while 10% of rats injected with transfectants containing mts-1 in anantisense orientation (cell lines 162/9 and 162/1) had metastases.

Hence transfection of a mammalian mts-1 gene into mice or rat cells cancause such cells to undergo metastasis when they are injected into amouse or rat.

EXAMPLE 14 CSML-100 Cells Grow More Slowly than CSML-0 Cells

Methods

CSML-0 and CSML-100 cells were seeded at a density of 10⁶ cells/dish andcounted the following day using a hemacytometer. The relative rates ofDNA synthesis were measured by incorporation of ³[H] thymidine. Bothexperiments were done in triplicate, and the data are reported as anaverage.

DNA synthesis was measured as follows. The cells were washed once withmedia. 2 mls of media containing 1 μl of ³[H] thymidine was added to thecells and incubated for 4 hours. The cells were washed twice with PBSand TCA precipitated following standard protocols. The TCA precipitatewas dissolved in 0.1N NaOH containing 0.5% Triton X-100 and placed onice for 30 minutes. The resultant suspension was added to 6 mls ofscintillation fluid for scintillation counting.

Results

Table 5 illustrates that less cell growth and less tritiated thymidineincorporation was observed for metastatic CSML-100 cells than fornon-metastatic CSML-0 cells.

TABLE 5 ³[H] Thymidine DNA Cell Line Incorporation (dpm) Cells/DishSynthesis/Cell CSML-0 6428 1.6 × 10⁶ 4.0 × 10⁻³ CSML-100 3700 1.1 × 10⁶3.3 × 10⁻³

In particular, only 1.1×10⁶ CSML-100 cells are observed per dish,whereas 1.6×10⁶ CSML-0 cells are observed. Since 1×10⁶ cells of bothtype were plated, the CSML-100 cell growth was only about one-sixth thatof the CSML-0 cell growth. FIG. 11 further illustrates that the growthof CSML-100 cells from a 2-day to at least a 5-day period is less thanthat of CSML-0 cells.

EXAMPLE 15 mts-1 mRNA can be Detected by Hybridization of mts-1Antisense Probes to Tissue Sections

Methods

Mouse embryonic trophoblast cells express mts-1. To illustrate theefficacy of mts-1 nucleic acid probes for detection of mts-1 mRNA intissue sections, frozen sections of an 8 day mouse embryo were obtained.Sections were placed onto a standard microscope slide and fixed for 5min. with 3% formaldehyde, 0.1M phosphate buffer, pH 7.2.

Sense and antisense mts-1 riboprobes were prepared by in vitrotranscription from a GEM-2-mts-1 vector containing the 3′untranslatedregion of mts-1 sing T₇ and T₃ RNA polymerases according to themanufacturers direction. Transcription was with ³H-UTP(45 μG, Amersham).

Prior to hybridization, slides were acetylated with 0.25% (v/v) aceticanhydride in 0.1M ariethanolamine for 10 minutes at room temperature.The sections were rinsed in 2×SSC and dehydrated through an ethanolseries (30%, 50%, 70%, 85%, 95%, 99%, 99%).

To the dried sections, 20 μl of hybridization solution (0.3M NaCl, 20 mMTris pH 8, 1 mM EDTA, 50% formamide, 10% dextran sulfate, 1×Denhardt's,500 μg/ml yeast RNA) containing about 10⁴ cpm/μl ³H sense or antisenseprobe, preheated to 80° C., was applied and secured with a coverslip.The slides were immersed in mineral oil and incubated at 45° C. forabout 12 hours. Excess mineral oil was removed from slides, and slideswere washed through chloroform 3 times. Slides were next rinsed3.times.5 in 4×SSC to remove coverslips. RNase digestion (20 μg/ml RNaseA, 1 U/ml RNase T₁) in 0.5M NaCl, 10 mM Tris pH 8.0, 1 mM EDTA) was donefor 30 minutes at 37° C. Slides were washed once in RNase buffer for 30minutes, 37° C.; then in 4 liters 2×SSC for 30 minutes at roomtemperature; next in 0.1×SSC at 55° C. for 30 minutes; finally in 4liters 0.1×SSC at room temperature for 30 minutes. Slides weredehydrated through an ethanol series containing 300 mM ammonium acetate.Once slides were dry, they were dipped in NTB-2 emulsion (Kodak) diluted1:1 with 600 mM ammonium acetate and autoradiographed for 4 weeks.Slides were developed in D-19 2.5 minutes, fixed in 2% acetic acid 30seconds, fixed for 5 minutes and rinsed in water for 30 minutes.Finally, sections were counter stained, cover slipped and photographedusing dark-field illumination.

Results

Hybridized mts-1 probe was detected in mouse trophoblast cells only whenthe mts-1 probe was an antisense probe (FIG. 12a). The sense mts-1 probegave rise to no signal (FIG. 12b). These data indicate that an antisensemts-1 probe can be used to detect mts-1 mRNA in tissue sections.

EXAMPLE 16 Chicken Anti-mts-1 Antibody Detects mts-1 Protein by WesternBlot and Immunohistochemistry

Methods

The chicken anti-mts-1 antibody (α-mts-1) was prepared as described inExample 9.

Lysates of CSML-0 and CSML-100 cells were electrophoresed on 12%SDS-PAGE gels and transferred to polyvinyledene difluoride (PVDF)membranes for Western blot analysis. Membranes containing 3 μg purifiedmts-1 were incubated with anti-mts-1 antibody (1:2000) for 3 hours atroom temperature and then with anti-chicken IgG-HRP at room temperaturefor 2 hours. Signal was detected with diaminobenzene (DAB).

To test the specificity of the α-mts-1 antibody, membranes containingCSML-100 proteins were probed with α-mts-1 in the presence and absenceof 260 ng free recombinant mts-1 protein. Membranes containing 3 μgpurified mts-1 were treated as above but upon addition of the primaryantibody (α-mts-1) 13 μg of purified free mts-1 was added.

Frozen mouse spleens were sectioned and fixed onto glass slides.Sections were probed with a 1:1000 dilution of α-mts-1 in PBA accordingto the method of Harlow et al. To test the specificity of the α-mts-1antibody, 130 ng free mts-1 protein was applied to one series of slidesalong with the diluted α-mts-1 antibody.

The α-mts-1 antibody was deemed specific for mts-1 protein when freemts-1 effectively eliminated binding of α-mts-1 to mts-1 Western blotsor mouse spleen sections.

Results

FIG. 13 depicts a Western blot of CSML-0 (Lane 1) and CSML-100 (Lanes 2and 3) cell lysates. Lanes 1 and 2 were probed with α-mts-1 antibodywithout added free mts-1 protein. As illustrated, a 10-12 Kd mts-1protein is expressed in CSML-100 cells (Lane 2) but not in CSML-0 cells(Lane 1). Moreover, 260 ng free mts-1 protein effectively eliminatedantibody binding to the CSML-100 cell lysate in Lane 3. Therefore, theα-mts-1 antibody is highly specific for mts-1 protein.

Similarly, mts-1 protein is detected in frozen mouse spleen sections(FIG. 14a) and α-mts-1 antibody binding on such tissue sections iseliminated when free mts-1 protein is applied to the sections with theantibody (FIG. 14b).

Accordingly, mts-1 protein can readily be detected on Western blots andon tissue sections using the α-mts-1 antibody.

EXAMPLE 17 Low Molecular Weight mts-1 Protein is Found Only in Serumfrom Animals with Metastatic Cancer

Methods

Mouse Studies: CSML-0 and CSML-100 cells were injected intravenouslyinto the tail. veins of A/J mice at 1×10⁵ cells per mouse (FIG. 15a,Lane 2) or 1×10⁶ cells per mouse (FIG. 15b, Lane 2). After three weeksthe mice were sacrificed, their lungs examined for the presence ofmetastasis, and their blood drawn. Blood was allowed to clot at roomtemperature for one hour and was then microfuged to isolate sera. Thesera samples were loaded at 100 μg per lane on a 13% SDS-PAGE gel (FIG.15a) or a 13% Tris-Tricine gel (FIG. 15b). The proteins were thentransferred to PVDF membranes and probed with a 1:1000 dilution ofα-mts-1 antibody and a horse-radish peroxidase conjugated secondaryantibody.

mts-1 is expressed in T lymphocytes and activated macrophages. To testwhether the detected mts-1 protein resulted from lysis of normal bloodcells including T lymphocytes and macrophages, whole mouse blood waslysed and probed with α-mts-1 antibody in a western blot analysis.

Whole blood was taken from a normal mouse, lysed in a triton-X100solution and electrophoresed on a 13% Tris-Tricine gel. A PVDF membraneblot of the gel was prepared and probed as above.

To determine whether the presence of mts-1 in sera is simply due to achronic immune response which might increase the number of T lymphocytesand activated macrophages, mice were injected with salmonella LPS overan extended time period to induce a chronic immune response. Sera weredrawn and western analysis was performed as described above.

Human Studies: Serum samples were obtained from normal women andpatients with breast carcinomas or advanced malignant lymphomas 150 μgof each serum sample was run on a 12% SDS-PAGE gel. The proteins weretransferred to PVDF membranes and the membranes were probed with a1:1000 dilution of α-mts-1 and then with a 1:1000 dilution of thesecondary antibody (rabbit anti-chicken IgG-HRP). The reaction wasdeveloped with a DAB solution.

Results

Mouse Studies: Three weeks after intravenous injection, the micereceiving CSML-100 looked very sick and had breathing difficulties.Western analysis of sera from injected and non-injected animalsindicated that only those mice receiving CSML-100 cells had a 10-12 Kdmts-1 protein (FIGS. 15a and 15 b, Lane 2). Injection of as little as10⁵ CSML-100 cells three weeks prior to western analysis produced apositive serum response. The lungs of those mice injected with either10⁵ or 10⁶ CSML-100 cells had extensive metastasis.

The α-mts-1 antibody detected a high molecular weight band in allsamples on the western blot. However, addition of free mts-1 protein tothe Western blot when incubating with the α-mts-1 antibody did noteliminate the signal from the high molecular weight band. Only the lowermolecular weight band found in CSML-100 injected mice was eliminated bycompeting free mts-1 protein. Therefore, only the lower molecular weightband is mts-1 protein. The higher molecular weight band may be anabundant serum protein which cross-reacts with the α-mts-1 polyclonalantibody.

FIG. 15c illustrates that the mts-1 protein detected in serum is not anormal component of whole blood and is not a result of a chronic immuneresponse. The mts-1 protein is not detected in lysed whole blood cells(FIG. 15c, Lanes 1-4 containing 5, 10, 20 and 25 μl lysed whole blood).

However, mts-1 was detected in similarly treated CSML-100 cells whichwere provided as a positive control (FIG. 15c, Lane 5).

FIG. 15d illustrates that the mts-1 protein detected in sera ofmetastatic cancer patients is not due to a chronic immune responseinduced by salmonella LPS over an extended period of time. The mts-1protein could not be detected in the 75 μg, 100 μg or 150 μg of serafrom chronically immunologically stimulated mice (FIG. 15d, Lanes 1-3).

Accordingly, a 10-12 Kd mts-1 protein can be detected in sera of micewith metastatic cancer. No mts-1 protein is detected in the serum ofcontrol mice.

Human Studies: FIG. 16 illustrates that mts-1 protein can be detectedonly in sera from patients known to have metastatic cancer. Anapproximate 27 Kd mts-1 protein could be detected in serum from apatient with metastatic breast cancer (FIG. 16, Lane 6) and in twopatients with metastatic lymphomas (FIG. 16, Lanes 5 and 7).

However, no such 27 Kd mts-1 protein was detected in serum from a normalpatient (FIG. 16, Lane 3) or in serum from patients with non-metastaticbreast cancer (FIG. 16, Lane 1) or non-metastatic lymphomas (FIG. 16,Lanes 2 and 4).

The higher molecular weight band apparent in FIGS. 16a-d is not mts-1protein. In particular, when the Western blot is probed with α-mts-1antibody in the presence of free mts-1 protein, only the 27 Kd proteinband disappears. Free mts-1 protein does not eliminate the highmolecular weight signal. Therefore, the α-mts-1 polyclonal antibody maycross-react with an abundant serum protein. Such cross-reactivity can beeliminated by, for example, using an antibody directed against humanmts-1 protein (α-mts-1 is directed against mouse mts-1 protein) or byemploying highly specific monoclonal antibodies prepared as described inExample 10.

Accordingly, mts-1 protein is detectable only in sera from patients withmetastatic cancer. The mts-1 protein cannot be detected in the serum ofnormal patients or in the serum of patients with non-metastatic cancer.Antibodies directed against mts-1 protein can therefore be used in asimple serum immunoassay to diagnose and detect metastatic cancer inpatients.

EXAMPLE 18 Materials and Methods

Plasmids

The coding region of mts1 was cloned in pSK3 (Pharmacia) vector,containing simian virus promoter. The resulting construct was named aspSV-mts1. In pHMG-mts1 the coding part of mts1 with the first intron wascloned in pHMG vector, containing the constitutive promoter ofhydroxymethyl-glutaryl-CoA-reductase (HMGCR). PSVBc12 was constructed bycloning the Bc12/Xhol insert from PEBS7-425 in pSK3 vector.

The following mouse wild type p53 PCRs were designed: #1—full sizecoding region (390 aa) was amplified using primers: forwardCGGGATCCGACTGGATGACTGCCATGGA (SEQ ID NO:10) (having a BamHI site),reverse CGAAGCTTCAGTCTGAGTCAGGCCCCACT (SEQ ID NO:11) (including aHindIII site); #2—N-terminal domain (106 aa): forward, same as theforward primer for #1, and reverse CGAAGTCTTGAAGCCATAGTTGCCCTGGTAAG (SEQID NO:12)(including a HindIII site); #3—DNA-binding domain (185 aa) :forward CGGGATCCCACCTGGGCTTCCTGCATGCT (SEQ ID NO:13) (including a BamHIsite), reverse CGAAGCTTGGACTTCCTTTTTTGCGGAAATTTTC (SEQ ID NO:14)(including a HindIII site); #4—C-terminal (99 aa): forwardCGGGATCCCTTTGCCCTGAACTGCCCCCA (SEQ ID NO:15) (including a BamHI site),and reverse—same as the reverse primer for #1. The PCR products weredigested with BamHI/HindIII and cloned in eukaryotic expression vectorpXmyctag, containing a CMV promoter and 8-aa myc tag, and bacterialexpression vector pQE30 (Qiagen). PSP65m65 plasmid DNA was used for theamplification of p53. Human pC53-SN3 (human wild type p53) and pC53-SCX3(human mutant Human mutant p53-pC53-SCX3 (143^(Val-Ala)) eukaryoticexpression plasmids were obtained. For conditional expression, mts1 cDNAwas excised, cloned in pUHD 10-3 and used for transfection of cell linesproducing reverse tetracycline-controlled transactivator (pUHD172-neo)(Clontech).

p21/WAF-luc was constructed by cloning 13 copies of p53 binding.consensus element from the p21/WAF promoter in the pfLUC reporterconstruct containing the Photinus pyralis luciferase gene under theminimal c-fos promoter Saksela et al. (Mol. Cell. Biol. 13:3698-3705,1993). The β-galactosidase expression plasmid was purchased fromClontech. pBabe-Hyg contains Hygromicin-resistance gene. pSV2-neocontains neomycin resistance gene.

Cell Lines and Transfection

Mouse mammary adenocarcinoma cell lines: CSML-0 and CSML-100 Senin etal. (Exp. Oncology USSR 5:35-39, 1983), VMR-liv Senin et al. (VestnikUSSR Acad. Med. Sci. 5:85-91, 1984) were derived from two independentspontaneous tumors in A/Sn mice. Saos-2 is a human osteosarcoma cellline.

Cells were transfected by electroporation: 1-3×10⁶ cells in 100 μl ofphosphate saline buffer were transferred into electroporation cuvetteand single pulse of 250V and 250 μFd was applied using Bio-Radelectroporation system. Clones were selected in the presence of 400 μgof G-418, for the conventional tetracyline inducible clones, doubleselection with G-418 and 200 μg/ml Hygromycin was used. In transienttransfection experiments, the efficiency of each transfection wasmonitored by use of a cotransfection of β-galactosidase expressionvector, pCMV-gal. At 24-48 hours posttransfection, cells were lysed andthe luciferase activity was measured with a luminometer (Promega Corp.).The same lysates were tested for β-galactosidase activity by usingo-nitrophenyl-β-galactopyranoside (Sigma) as a chromogenic substrate.

Preparation of Recombinant Proteins

Histidine-tagged p53 and Mts1 proteins were expressed in XL-blueEscherichia coli by induction with 0.2 mM isopropylβ-D-thiogalactopyranoside for 4 hours at 37° C. Protein isolation indenaturating conditions followed by renaturation were performedaccording to the manufacturer's protocol (Qiagen).

Western Blotting

Protein isolation and western blotting were performed according toGrigorian et al. (Int. J. Cancer 67:831-841, 1996) (IJC). Immunostainingand protein bands visualization with ECL system SuperSignal® (Pierce)were carried out according to the manufacturer's protocol.

Indirect Immunoprecipitations and in vitro Pull-down Assay

Cells were metabolically labeled for 4 h in methionine-cystein-freemedium supplemented with dialyzed and inactivated 10% FCS with 0.2mCi/ml [³⁵S]-methionine and -cystein (Amersham). The cells were lysed in150 mM NaCl-50 mM Tris-Hcl pH 7.6 -0.5% NP-40 and precleaned on 50%protein A-Sepharose. The precleaned lysates were incubated for 2 hourswith anti-p53 antibodies: monoclonal pAb421 and goat polyclonal E-19(Santa Cruz Biotechnology, Inc.) and anti-Mts1 rabbit serum, followed by5 washes with the same buffer. The precipitated proteins were separatedon gradient 4-20% PAAG and detected by autoradiography.

For in vitro pull down assay, 1 μg recombinant Mts1 was mixed withrecombinant full size p53 and its domain peptides in 150 mM NaCl-50 mMTris-HCl pH 8.0-0.5% NP-40 and precleaned on Protein A-Sepharose on thepresence of protease inhibitors at 1 hour in cold room. To theprecleaned mixtures, fresh portions of the protein A-sepharose and thecorresponding anti-p53 antibodies were added: pAb421 for full-size andC-terminal domain, pAb240 for DNA-binding core domain and E-19 for theN-terminal domain, and incubated for 2 hours in the cold room. Following5 washes, immunoprecipitates were denaturated by heating at 100° C.-5min, separated in 15% PAAG and transferred to Immobilon-P (Millipore).To detect the co-immunoprecipitated Mts1 protein, membranes were probedwith anti-Mts1 antibody and developed by the ECL System. Recombinanthuman wild type GST-p53 and GST-p53-Δ30 (deletion mutant lacking aminoacid residues 364-393) fusion proteins were used for pull downexperiments with the Mts1 recombinant protein. 5 μg of GST andGST-fusion proteins coupled with Glutathione-sepharose beads wereincubated with 2 μg of the Mts1 protein in NP-40 buffer (1% NP-40, 50 mMTris-HCl pH 8.0-150 mM NaCl) for 2 h in the cold room with rotation.Beads with proteins bound were washed 5 times with NP-40-buffer.Proteins were isolated by boiling in the protein loading buffer for 5min and analyzed using Western blotting.

Phosphorylation Assays

Reactions were performed in a mixture (2 μl) containing 50 mM Tris-HClpH 7.6, 0.2 M NaCl, 10 mM MgCl₂, 4 mM Cacl₂, 2 mM dithiothreitol, 15 μlATP (Amersham Pharmacia Biotech), 25 μCi [γ-³²P]-ATP (5000 Ci/nnol,Amersham Pharmacia Biotech), 1 μM recombinant wild type p53 or thisprotein fragments for 30 min at 30° C. PKC assay was done in thepresence of 7.5 μg of phosphatidylserine (Sigma) by 0.025 μg PKC(Roche). CKII was purchased from New England BioLabs Inc., and 50 unitswere applied per each reaction. Recombinant Mts1 was sued inconcentrations of 3,5 and 9 μM reactions were terminated by 15%SDS-PAGE. Gels were fixed in 10% trichloracetic acid, dried and exposedto Kodak x-ray film.

Electromobility Shift Assay (EMSA)

Nuclear extracts were prepared as previously described by Kustikova etal. (Mol. Cell Biol. 12:7095, 1998). To perform EMSA, nuclear extractswere incubated with end-labeled oligonucleotides that contained bindingsites for p53 or Oct-1 proteins. Oligonucleotide sequences were asfollows: for oct-1-TGCGAATGCAAATCACTAGAA (SEQ ID NO:16) (LeBowitz J. H.,Genes Dev. 2, 1227-1237, 1998); for p53-GAACATGTCCCAACATGTTG (SEQ IDNO:17), derived from the promoter of p21/WAF Avantaggiati et al. (Cell89:1175-1184, 1997). The reactions were carried out in 10 μl of thebuffer containing 100 mM KCl, 1 mM MgCl₂, 1 mM DTT, 0.1% NP-40, 0.5mg/ml BSA, 5% glycerol. To perform gel supershift analysis, anti-p53antibody (pAb421) were added to the EMSA reaction mixtures. Theincubation with antibody was carried out for 1 h at 4° C. after thebinding reactions were completed.

Northern Blot Analysis

CSML-0 conventional Mts1-tet-inducible clones were grown at low anddense conditions and induced with 2 μg/ml Doxycylin at 0.24,48 and 72 h.RNA was isolated according to Chomczynski et al. (Anal. Biochem.162:156-159, 1987). Gel elecrophoresis and Northern blot analyses wereperformed as it is described in Grigorian et al. (Int. J. Cancer67:831-841, 1996). The filters were sequentially hybridized with murinep21/WAF, Bax and Cyclin G1 probes. The amounts of mRNA on the filterswere calibrated by hybridization with γ-³²P-ATP-labeled poly(U) probe.To quantify the intensities of the bands membranes were scanned using aMolecular Dynamics computing densitometer (Sunnyvale, Calif.) withImageQuant software, after each hybridization.

EXAMPLE 19 MTS-1 Binds to the C-Terminal Domain of P53

To determine whether Mts-1 and p53 proteins directly interact with eachother, immunoprecipitation(IP) and Far Western experiments wereperformed. In these experiments, two cell lines were used: CSML-0 cellswhich express very low level of wt-p53 and does not express Mts1 at all,and CSML100 cells which express mutant p53 and high level of Mts1.

CSML-0 were transfected with tet-inducible Mts1. Lysates frommetabolically labeled cells were used for IP with anti-p53 and anti-Mts1antibodies. As shown in the IP—radioautography assays following SDS-PAGEelectrophoresis (FIG. 17), the p53 protein band was readily detected inanti-Mts1 immunoprecipitates and, vice versa, the Mts1 protein band inanti-p53 immunoprecipitates. As a positive control, the bandcorresponding to the heavy chain of the non-muscle myosin, a knowntarget of Mts1, was also detected in the anti-Mts1 immunoprecipitates.

Non-radioactive IP assays with the lysate obtained from 1×10⁹ CSML-100cells were-performed using several anti-p53 antibodies targetingdifferent epitopes located at N-terminal and C-terminal domains of p53.Immunoprecipitates were subjected to Western blot analysis and probedwith anti-Mts1 antibody. The Mts1 protein was easily detected in thecomplex precipitated by pE19 antibody, directed to the N-terminal domainof p53. The Mts1 protein was not detected in the complexes precipitatedby antibodies against C-terminal domain of p53, pAb421 and p122Ab (FIG.18).

To identify the domain of p53 that interacts with Mts1, the recombinantp53 domains corresponding to N-terminal (transactivation) AA 1-106, core(DNA-binding) AA 104-288 and C-terminal (oligomerization and regulation)AA 289-387 were obtained. Full-size p53 and above mentioned domains wereco-immunoprecipated with Mts1 recombinant proteins by anti-p53 antibody.Immunoprecipitates were subjected to Western blot analysis usinganti-mts1 antibody. As is shown in FIG. 19, only full-size p53 and theC-terminal domain of p53 immunoprecipitated the Mts1 protein, but notthe N-terminal and the DNA-binding domains.

To more precisely map the interaction site, the recombinant wt-p53-GSTand Δ30p53-GST (mutant p53 lacking amino acids 364-393 of the C-terminaldomain), captured on the Glutathione-Sepharose4B beads, were incubatedwith recombinant Mts1. Mts proteins bound to wild type or mutant p53were recovered in SDS-protein loading buffer by boiling followed byPAGE, Western blotting and immunoprobing with anti-Mts1 antibody. FIG.20 illustrates that p53 molecule with deletion in the C-terminal domainwas not able to bind the Mts1 protein, and thus, that the binding siteis spread along 364-393 aa of p53.

Another approach, Far-Western blot analysis, was also employed to assessthe interaction between Mts1 and p53. Full size p53 and its functionaldomains, expressed in E.coli, were separated on SDS-PAGE and transferredinto Immobilon-P. Filters were incubated with recombinant Mts1 inconditions allowing the interaction with the proteins fixed on themembrane. Mts1 bound to p53 proteins on the filter, was detected withanti-Mts1 antibody. Data shown in FIG. 21, consistent with the IPresults, indicated that Mts1 was able to bind full-size p53 and itsC-terminal domain. As a positive control we have used recombinantfragment of non-muscle myosin which is known as a target for Mts1protein (FIG. 21, lane 5). BSA loaded in 5× excess did not revealnonspecific mts1 binding in Far-Western assay, neither did N-terminal orDNA-binding domains.

EXAMPLE 20 MTS1 Protein Inhibits Phosphorylation of P53 by PKC

The C-terminal domain of p53 contains PKC and CKII phosphorylationsites. Experiments were carried out to determine whether Mts1 affectsthe phosphorylation of p53. One micromolar recombinant full-size p53 andits distinct domains were phosphorylated by PKC in the absence andpresence of 3, 5 and 9 μM recombinant Mts1 protein, respectively, andsubsequently analyzed by SDS-PAGE.

As shown in FIG. 22, Mts-1 inhibited the phosphorylation of full-sizep53 and the C-terminal protein fragment by PKC. Addition of the sameconcentrations of Mts1 to the PKC reaction mixture did not affect thephosphorylation of the N-terminal and DNA-binding domains of p53. Nointerference of Mts1 was shown with CK II phosphorylation of p53 and itsdomains (FIG. 23). These observations indicate that Mts1 specificallyinhibited the phosphorylation of PKC of the C-terminal domain of p53.

EXAMPLE 21 Inhibition of P53 DNA-binding Activity by MTS1

Effects of the Mts1-p53 interaction on the DNA-binding activity of p53were investigated in an electrophoretic mobility shift assay (EMSA)(FIG. 24).

The end-labeled oligonucleotide containing p53-binding site from p21/WAFpromoter was mixed with nuclear extracts containing wild typep53(lanes_(—)1). Specificity of the DNA-protein complexes was confirmedby supershift of the complexes after adding anti-p53 antibody (lanes 7),competition with specific p21/WAF p53 binding site-containingoligonucleotide (lanes 2-5) and absence of influence of non-specificnucleotide (lanes 6,14-16). Incubation of nuclear extracts with the Mts1protein before adding the labeled oligonucleotide decreased DNA-bindingactivity of p53 in dose-dependent manner (lanes 8-10). The inhibitionwas less when Mts1 was added after formation of p53-DNA complexes. Mts1did not affect the binding Oct-1 factor to oligonucleotide containingOct-1 binding site (lanes 15).

These results indicate that Mts-1 inhibited the DNA-binding activity ofp53.

EXAMPLE 22 MTS1 Affects P53-Dependent Transcription

Mts1 cDNA was placed under the control of HMGCG promoter and wascotransfected with constructs bearing p53-binding sites from the p21/WAFpromoter, fused with the luciferase reporter gene. Three differentmts1-negative cell lines, mouse low (VMR-liv), nonmetastatic (CSML-0)adenocarcinoma cell lines with wtp53 and p53-null Saos-2.1109 cells,were used in this assay. A pCMV-β-gal plasmid was cotransfected forevaluation of the transfection efficiency and the degree of apoptosis.Luciferase activity was adjusted by β-galactosidase activity in eachtransfection. The results of the 2-3 experiments are summarized in FIGS.25A and 25B.

The results indicated that Mts1 affected the transactivation activity ofp53. In all analyzed cell lines, the presence of Mts1 correlated withthe inhibition of the luciferase activity transcribed from the promotercontaining the p21/WAF p53-binding consensus. The inhibition was 2-3folds in CSML-0 and VMR-liv, and 1.3-1.4 fold in Saos-2.1109 cells.

EXAMPLE 23 Anti-MTS1 Antibody Enhances Tumor Necrosis and InhibitsMetastasis

Mice bearing highly metastatic CSML-100 tumors were treated withanti-Mts1 antibody from the initial stages of tumor development via i/vinjections. In 4 weeks animals were sacrificed. Tumors, lungs and liverswere subjected to histological analysis (FIG. 26). Large necrotic areaswere observed in tumor mass treated with anti-Mts1 antibody (A,right)compared to tumors treated with control IgG (A,left).

Decrease of metastases was observed in lungs among animals treated withanti-Mts1 antibody (B,C) compared to control animals (D,E).

One experiment with 4 animals in each group was done.

17 1 303 DNA Homo sapiens 1 atggcgtgcc ctctggagaa ggccctggat gtgatggtgtccaccttcca caagtactcg 60 ggcaaagagg gtgacaagtt caagctcaac aagtcagagctaaaggagct gctgacccgg 120 gagctgccca gcttcttggg gaaaaggaca gatgaagctgctttccagaa gctgatgagc 180 aacttggaca gcaacaggga caacgaggtg gacttccaagagtactgtgt cttcctgtcc 240 tgcatcgcca tgatgtgtaa cgaattcttt gaaggcttcccagataagca gcccaggaag 300 aaa 303 2 101 PRT Homo sapiens 2 Met Ala CysPro Leu Glu Lys Ala Leu Asp Val Met Val Ser Thr Phe 1 5 10 15 His LysTyr Ser Gly Lys Glu Gly Asp Lys Phe Lys Leu Asn Lys Ser 20 25 30 Glu LeuLys Glu Leu Leu Thr Arg Glu Leu Pro Ser Phe Leu Gly Lys 35 40 45 Arg ThrAsp Glu Ala Ala Phe Gln Lys Leu Met Ser Asn Leu Asp Ser 50 55 60 Asn ArgAsp Asn Glu Val Asp Phe Gln Glu Tyr Cys Val Phe Leu Ser 65 70 75 80 CysIle Ala Met Met Cys Asn Glu Phe Phe Glu Gly Phe Pro Asp Lys 85 90 95 GlnPro Arg Lys Lys 100 3 579 DNA Homo sapiens 3 ggcagttgag gcaggagacatcaagagagt atttgtgccc tcctcgggtt ttaccttcca 60 gccgagattc ttcccctctctacaaccctc tctcctcagc gcttcttctt tcttggtttg 120 atcctgactg ctgtcatggcgtgccctctg gagaaggccc tggatgtgat ggtgtccacc 180 ttccacaagt actcgggcaaagagggtgac aagttcaagc tcaacaagtc agaactaaag 240 gagctgctga cccgggagctgcccagcttc ttggggaaaa ggacagatga agctgctttc 300 cagaagctga tgagcaacttggacagcaac agggacaacg aggtggactt ccaagagtac 360 tgtgtcttcc tgtcctgcatcgccatgatg tgtaacgaat tctttgaagg cttcccagat 420 aagcagccca ggaagaaatgaaaactcctc tgatgtggtt ggggggtctg ccagctgggg 480 ccctccctgt cgccagtgggcacttttttt tttccaccct gctccttcag gacacgtgct 540 tgatgctgag caagttcaataaagattctt ggaagttta 579 4 10 PRT Artificial Sequence Description ofArtificial Sequencepeptide 4 Ala Cys Pro Leu Glu Lys Ala Leu Asp Val 1 510 5 16 PRT Artificial Sequence Description of ArtificialSequencepeptide 5 Lys Glu Gly Asp Lys Phe Lys Leu Asn Lys Ser Glu LeuLys Glu Leu 1 5 10 15 6 13 PRT Artificial Sequence Description ofArtificial Sequencepeptide 6 Leu Pro Ser Phe Leu Gly Lys Arg Thr Asp GluAla Ala 1 5 10 7 15 PRT Artificial Sequence Description of ArtificialSequencepeptide 7 Asn Glu Phe Phe Glu Gly Phe Pro Asp Lys Gln Pro ArgLys Lys 1 5 10 15 8 21 DNA Artificial Sequence Description of ArtificialSequenceprimer 8 atggcgtgcc ctctggagaa g 21 9 21 DNA Artificial SequenceDescription of Artificial Sequenceprimer 9 tttcttcctg ggctgcttat g 21 1028 DNA Artificial Sequence Description of Artificial Sequenceprimer 10cgggatccga ctggatgact gccatgga 28 11 29 DNA Artificial SequenceDescription of Artificial Sequenceprimer 11 cgaagcttca gtctgagtcaggccccact 29 12 32 DNA Artificial Sequence Description of ArtificialSequenceprimer 12 cgaagtcttg aagccatagt tgccctggta ag 32 13 29 DNAArtificial Sequence Description of Artificial Sequenceprimer 13cgggatccca cctgggcttc ctgcatgct 29 14 34 DNA Artificial SequenceDescription of Artificial Sequenceprimer 14 cgaagcttgg acttccttttttgcggaaat tttc 34 15 29 DNA Artificial Sequence Description ofArtificial Sequenceprimer 15 cgggatccct ttgccctgaa ctgccccca 29 16 21DNA Artificial Sequence Description of Artificial Sequenceprimer 16tgcgaatgca aatcactaga a 21 17 20 DNA Artificial Sequence Description ofArtificial Sequenceprimer 17 gaacatgtcc caacatgttg 20

What is claimed is:
 1. A method of treating a cancer in a subjectwherein mts-1 is expressed in the cancerous cells, comprisingadministering to said subject an antibody reactive with said mts-1protein.
 2. The method of claim 1 wherein said antibody is conjugated toa toxin.
 3. A method of inactivating, destroying, or nullifying an mts-1protein or cells expressing the mts1 protein comprising directingantibodies against said mts1 protein.
 4. The method of claim 3, whereinsaid mts-1 protein is expressed by metastatic cancer cells in a subject.5. The method of claim 1 or 4 wherein said cancer is selected from thegroup consisting of lung, liver, kidney, thyroid, breast, leukemic,pancreatic, endometrial, ovarian, cervical, skin, colon or lymphoidcancer.